bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 Title:

2 Identifying dynamic protein and RNA proximity interaction networks of actinin reveals RNA-

3 binding and metabolic regulatory mechanisms

4

5 Feria A. Ladha M.S.1, Ketan Thakar Ph.D.2*, Anthony M. Pettinato B.S.1*, Nicholas Legere

2 2 1 1 2 6 B.S. , Rachel Cohn M.S. , Robert Romano B.S. , Emily Meredith B.S. , Yu-Sheng Chen Ph.D. ,

7 and J. Travis Hinson M.D.1,2,3

8 Affiliations:

9 1University of Connecticut Health Center, Farmington, CT 06030, USA

10 2The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA

11 3Cardiology Center, UConn Health, Farmington, CT 06030, USA

12 *These authors contributed equally

13

14 Lead Contact: Correspondence: [email protected]

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2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 Abstract

2 Actinins are actin cross-linkers that are expressed in nearly all cells and harbor mutations in

3 heritable diseases. We deployed proximity-dependent biotinylation to identify actinin proximity

4 proteins and RNA transcripts in human cardiomyocytes to reveal new functions of the actin

5 cytoskeleton. We identified 285 proximity protein partners including unexpected effectors of

6 RNA-binding and metabolism, and interrogated dynamic partners using a sarcomere assembly

7 model. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein

8 associated with type 2 diabetes, interacted with the rod domain of actinin through its K

9 Homology domain. This interaction was necessary for actinin localization and translation of

10 electron transport chain transcripts, and oxidative phosphorylation. Diminished IGF2BP2 in

11 cardiac microtissues impaired contractile function in accord with diminished metabolic functions.

12 This actinin proximity protein and RNA study expands our functional knowledge of the actin

13 cytoskeleton, and uncovers new modes of metabolic regulation through interactions with RNA-

14 binding proteins.

15

16 Main

17 Actinin proteins are ubiquitous spectrin family members that cross-link actin, and are important

18 for a myriad of cellular functions including cell adhesion, migration, contraction, and signaling1.

19 The four human actinin isoforms have distinct expression profiles (non-muscle ACTN1 and

20 ACTN4; cardiac muscle ACTN2; and skeletal muscle ACTN3), but share homologous amino

21 acid sequences that are organized into three structural domains—an actin-binding (AB) domain

22 composed of two calponin-homology domains, a central rod domain containing four spectrin-like

23 repeats (SR), and a calmodulin-like domain (CaM) containing two EF hand-like motifs2. While it

24 is thought that actinin evolved initially to regulate the early eukaryotic actin-based cytoskeleton3,

25 it has acquired more elaborate functions in vertebrates, including a mechanical role in the

26 sarcomere, a specialized contractile system required for striated muscle function3, 4. Moreover,

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 normal actinin function is critical for multiple human organ systems as inheritance of ACTN1,

2 ACTN2, and ACTN4 mutations have also been linked to congenital macrothrombocytopenia,

3 hypertrophic cardiomyopathy, and focal segmental glomerulosclerosis of the kidney,

4 respectively5-7.

5 In the cardiac sarcomere, alpha actinin-2 (referred henceforth as actinin) is the major

6 structural component of the Z-disk, where it is essential for sarcomere assembly and function8.

7 Actinin regulates sarcomere assembly by providing a scaffold for protein-protein interactions

8 (PPIs) such as with titin (TTN) and actin through CaM and AB domains, respectively8-10.

9 Secondary to these interactions, the thin and thick filament sarcomere systems are organized to

10 promote twitch contraction. It has also been observed that while the Z-disk is one of the stiffest

11 sarcomere structures, actinin demonstrates remarkably dynamic and complex molecular

12 interactions such as with the actin cytoskeleton that are necessary for normal myocyte

13 function11. Moreover, actinin interaction partners have also been described to exit the

14 sarcomere to execute critical roles in mechanotransduction signaling, protein homeostasis and

15 transcriptional regulation12. Secondary to the vast repertoire and dynamics of actinin molecular

16 partners, there remain extensive gaps in our knowledge of not only how actinin regulates

17 sarcomere assembly and function, but also within other cellular contexts enriched for actinin

18 expression such as at focal adhesions and cell surface receptors in both striated and non-

19 striated cells that may inform how actinin mutations cause human disease13, 14.

20 Here, we comprehensively identified the actinin protein interactome in CRISPR/Cas9-

21 engineered human cardiomyocytes using proximity-dependent biotinylation (BioID). This study

22 utilizes cardiomyocytes (iPSC-CMs) differentiated from induced pluripotent stem cells (iPSCs)

23 that express actinin fused to the promiscuous biotinylating enzyme BirA* from the ACTN2 locus

24 to provide in vivo actinin expression levels and localization15. We identify a high-confidence list

25 of actinin neighborhood proteins including those that are dynamically regulated by sarcomere

26 assembly to reveal molecular insights into this process as well other cytoskeletal functions. Our

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 study uncovers unexpected actinin interactions with a cluster of RNA-binding proteins including

2 IGF2BP2, which enabled us to study RNA neighborhoods in actinin proximity, and to uncover

3 new crosstalk between the actin cytoskeleton and oxidative phosphorylation that we find critical

4 to cardiomyocyte contractile function. Our study also provides a list of molecules that can be

5 studied to reveal insights into actin cytoskeletal dynamics and unexpected functions of actinin.

6

7 Results

8 BioID to identify actinin proximity partners

9 Actinin is an integral member of the actin cytoskeleton where it interacts within multiprotein

10 complexes with diverse biophysical properties such as within Z-disks and membrane-associated

11 focal adhesions, which are also dynamically regulated (Fig. 1a). Secondary to these actinin

12 properties, we selected the method of BioID that has been shown to be effective within these

13 contexts16. BioID uses BirA*, which is a genetically-modified version of E. coli BirA that enables

14 promiscuous lysine biotinylation within ~10 nm radius of expression (Fig. 1b)17.

15 We employed a genome engineering strategy to fuse BirA* with a hemagglutinin (HA)

16 epitope tag to the carboxyl-terminus of endogenous ACTN2 (Actinin-BirA*) in a human iPSC line

17 using CRISPR/Cas9-facilitated homologous recombination (extended data Fig. 1a). We then

18 selected and expanded an iPSC clone that expressed Actinin-BirA* fusion protein in directly-

19 differentiated iPSC-CMs (Fig. 1c)18, 19. To first verify that Actinin-BirA* functioned similarly to

20 unmodified actinin, we studied 3-dimensional cardiac microtissue assays that we previously

21 utilized to test variants in sarcomere genes that cause heart failure20, 21. Unlike cardiac

22 microtissues with heart failure-associated mutations, twitch force was unaltered by Actinin-BirA*

23 expression (extended data Fig. 1b). We confirmed appropriate localization of Actinin-BirA* to the

24 Z-disk by both co-localization immunofluorescence (Fig.1d) and HA-immunoprecipitation assays

25 (Fig.1e). Actinin-BirA* interacted with the Z-disk protein titin-cap (TCAP), but not the sarcomere

26 M-line protein myomesin22-25, which supported appropriate localization. With the knowledge that

5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 Actinin-BirA* functions similarly to unmodified actinin, we next optimized biotin supplementation

2 conditions in iPSC-CMs and confirmed that optimal labeling is achieved after 50 μM biotin

3 supplementation for 24 hours (Fig.1f, extended data Fig. 1c). Under these conditions, actinin-

4 BirA* biotinylated targets that overlapped actinin localization in iPSC-CMs (Fig.1d), and

5 demonstrated abundant protein biotinylation compared to controls (Fig. 1g).

6 To identify and quantify actinin proximity proteins, we subjected both biotin-pulsed

7 control and Actinin-BirA* iPSC-CM lysates to streptavidin immunoprecipitation and tandem

8 mass tags (TMT) quantitative proteomics (Fig. 1f). TMT of Actinin-BirA*- and control-precipitated

9 proteins analyzed using hierarchical clustering and heatmap analysis (Fig.1h) demonstrated

10 both low inter-replicate variability, and 285 high-confidence actinin proximity partners (extended

11 data Table 1 and 2). The proximity list was next examined using Search Tool for the Retrieval of

12 Interacting Genes/Proteins (STRING)26 network analysis (extended data Fig. 1d). STRING

13 confirmed identification of previously-known direct actinin interaction partners localized to the Z-

14 disk including TTN10, TCAP10 and myozenin 2 (MYOZ2)27 (Fig. 1i), which also belonged to a

15 sarcomere cluster including numerous other sarcomere components determined by Gene-

16 Ontology (GO) enrichment analysis. Actinin network proteins were also enriched within a focal

17 adhesion cluster including previously-known direct actinin partner zyxin (ZYX)28 (Fig.1j), and a

18 cell junction (Fig. 1k) cluster that includes the previously-known actinin direct partner catenin

19 alpha 1 (CTNNA1)29. BioID also uncovered less well-described actinin proximity network

20 clusters including metabolism (Fig. 1l), mitochondrial membrane (Fig. 1m) and RNA-binding

21 (Fig. 1n).

22 Actinin proximity partners change with sarcomere assembly

23 During sarcomere assembly, actinin first organizes into punctate Z-bodies, or pre-myofibrils, that

24 coalesce to form striated Z-disks30, and this assembly process, though critical to cardiomyocyte

25 contractile function, is incompletely understood. As it has been previously observed that

26 cardiomyocytes that lack troponin T (cTnT; encoded by TNNT2) only assemble Z-bodies31, we

6 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 studied cTnT knockout (KO) and wildtype (WT) iPSC-CMs as a model to understand actinin

2 proximity partners through two stages of sarcomere assembly. We started by inserting a

3 homozygous cTnT-KO mutation into the Actinin-BirA* iPSC line using CRISPR/Cas9 and error-

4 prone non-homologous end joining repair. We verified that cTnT-KO iPSC-CMs expressed no

5 cTnT protein (extended data Fig. 2a), and actinin antibodies only decorated Z-body structures

6 (Fig. 2a). We then performed TMT, and compared actinin proximity partners in cTnT-WT to

7 cTnT-KO iPSC-CMs (extended data Fig. 2b). Overall, sarcomere assembly enhanced actinin

8 interactions as we observed an increase in the relative intensity values for cTnT-WT relative to

9 both cTnT-KO and control samples (Fig. 2b). Next, we examined actinin proximity partners by

10 their dependence on sarcomere assembly (Fig. 2c, extended data Table 3). We first tested a

11 prevailing sarcomere assembly hypothesis that non-muscle myosin isoforms localize to Z-

12 bodies, and are subsequently replaced by muscle myosin isoforms in Z-disks30. Our results are

13 consistent with this model as actinin proximity with cardiac muscle beta isoform (MYH7)

14 increased by 89% with Z-disk assembly in cTnT-WT iPSC-CMs, while non-muscle type B

15 isoform (MYH10) exhibited a negligible 4% increase. Overall, we identified 80 sarcomere

16 assembly-dependent actinin proximity partners that clustered into GO terms for “sarcomere”,

17 “unfolded protein response”, “translation”, “glycolysis” and “mitochondrial membrane” (Fig. 2d).

18 Sarcomere assembly-dependent actinin interactions also demonstrated a shift from

19 proximity partners localized in focal adhesions to the sarcomere. For example, the most

20 sarcomere assembly-dependent enrichment partner (123% increase) was cysteine and glycine

21 rich protein 3 (CSRP3) (Fig. 2c). CSRP3 is a Z-disk protein that has been implicated to regulate

22 mechanical stretch signaling32, anchor calcineurin to the sarcomere33, and cause heart failure

23 when mutated34. In contrast, focal adhesion components including talin 2 (TLN2) were depleted

24 (12% decrease) from actinin proximity (Fig. 2c). Sarcomere assembly also enhanced actinin

25 proximity partners with metabolic functions and with mitochondrial membrane localizations,

26 which suggested that spatial regulation of these co-localized modules may be important in

7 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 metabolic regulation (Fig. 2d). In support of this, sarcomere assembly increased mitochondrial

2 content (extended data Fig. 2c) and oxygen consumption rates (extended data Fig. 2d, 2e). In

3 summary, sarcomere assembly shifted actinin proximity partners from focal adhesions to the

4 sarcomere; differentially-regulated muscle and non-muscle myosins; co-localized mitochondrial

5 membrane, metabolic and translational factors; and increased oxidative metabolism.

6 RIP-seq to identify actinin proximity RNA transcripts

7 We next sought to identify actinin proximity RNA transcripts that could reveal insights into new

8 actinin functions in RNA biology. With new knowledge of actinin proximity partners that bind

9 RNA (Fig.1n), and with Actinin-BirA* iPSC-CMs that could enable indirect RNA transcript

10 precipitation through affinity purification of biotinylated RNA-binding proteins using streptavidin,

11 we performed a modified version of RNA immunoprecipitation followed by sequencing (RIP-seq)

12 (Fig. 3a)35. Principle component analysis (PCA) of RIP-seq data demonstrated distinct clustering

13 of Actinin-BirA* samples compared to controls (Fig. 3b), and differential expression analysis

14 identified 945 transcripts that were actinin proximity-enriched. GO analysis (Fig. 3c) revealed

15 overwhelming enrichment of transcripts encoding electron transport chain (ETC) proteins (Fig.

16 3d, extended data Fig. 3a). In contrast, we found no actinin enrichment for glycolysis transcripts

17 (Fig. 4e, extended data Fig. 3b). 1113 transcripts were actinin proximity-depleted, while GO

18 terms revealed functions in chromatin and the nucleus (Fig. 3c). Cytoskeletal transcripts

19 including those encoding sarcomere thin and thick filament proteins were surprisingly depleted

20 (Fig.3f, extended data Fig. 3c) from actinin proximity despite their proteomic enrichment.

21 Following qPCR validation of RIP-seq hits (extended data Fig. 3d), we performed RNA

22 fluorescence in situ hybridization (RNA FISH) to confirm ETC transcript localization relative to

23 actinin protein expression. Actinin protein co-localized with transcripts encoding NDUFA1, an

24 ETC complex I component, but not TTN (Fig. 3g, 3h). We also validated two additional actinin

25 protein-enriched transcripts including NDUFA8 and GPX4 (Fig. 3h). We next assessed for

26 NDUFA1 and NDUFA8 transcript co-localization, which could reflect ETC transcript storage or

8 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 processing mechanisms. While both NDUFA1 and NDUFA8 transcripts localized near actinin

2 protein, they did not overlap (extended data Fig. 3e).

3 With knowledge of actinin-localized transcripts, we next performed polysome profiling

4 (Fig. 3i) followed by sequencing (Polysome-seq)36 to understand if actinin proximity localization

5 related to translation. PCA analysis demonstrated tight clustering of replicates of polysome-

6 bound transcripts compared to controls (Fig. 3j), and 273 transcripts were shared between the

7 polysome- and actinin protein-enriched fractions (Fig. 3k). Shared transcripts represented

8 28.9% of total actinin protein-enriched transcripts, and 24.7% of total polysome-enriched

9 transcripts. GO term analysis of overlapped transcripts revealed mitochondrial functions, which

10 was particularly prominent for ETC complex members (Fig. 3l, extended data Fig. 3f).

11 Conversely, actinin proximity-depleted but polysome-enriched transcripts enriched for distinct

12 GO terms including ribosome and troponin complex (Fig. 3l, extended data Fig. 3g).Together,

13 these data provide evidence that actinin functions as a PPI hub by co-localizing mitochondrial

14 membrane, RNA-binding, and translational proteins with transcripts involved in oxidative

15 phosphorylation.

16 IGF2BP2 binds actinin rod domains through K homology domains

17 Direct actinin interactions with RNA-binding proteins have not been previously well-described,

18 but this knowledge could provide new mechanisms of RNA regulation. Because ETC transcripts

19 were in proximity to actinin, we focused our study on candidate actinin-interacting RNA-binding

20 proteins secondary to their association with metabolic functions in the literature including

21 IGF2BP237, PCBP138, PCBP239, and SERBP140. We first confirmed actinin proximity results

22 using streptavidin affinity purification followed by immunoblotting against the four RNA-binding

23 proteins (Fig. 4a, extended data Fig. 4a). We also co-immunoprecipitated Actinin-BirA* using an

24 HA epitope antibody and found supportive evidence that IGF2BP2, PCBP1 and PCBP2

25 physically interacts with actinin (Fig. 4b, extended data Fig. 4b). The lack of SERBP1 co-

26 immunoprecipitation suggested a more complex interaction mechanism.

9 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 To extend co-immunoprecipitation results, we also tested the mode of interaction

2 between actinin and IGF2BP2, PCBP1, PCPB2, and SERBP1 using mammalian 2-hybrid (M2H)

3 assays. We began by constructing expression vectors encoding full-length or structural domains

4 of actinin and RNA-binding proteins. M2H using IGF2BP2 as “prey” and full-length actinin as

5 “bait” resulted in luciferase activation, but not for PCBP1, PCBP2 or SERBP1 (Fig. 4c, extended

6 data Fig. 4c). We expanded our M2H study to evaluate other actinin isoforms (ACTN1-4), and

7 all muscle and non-muscle isoforms interacted with IGF2BP2 illustrating that this interaction is a

8 general feature of all actinins (extended data Fig. 4e). We next mapped the IGF2BP2-actinin

9 interaction to IGF2BP2’s K Homology (KH) structural domain but not its RNA recognition motif

10 (RRM). Using IGF2BP2 as “bait”, we then mapped the actinin-binding site to its rod domain (Fig.

11 4d). In support of these findings, we observed co-localization of IGF2BP2 with actinin by

12 immunofluorescence, which was especially prominent at Z-disks (Fig. 4e). These data

13 (extended data Fig. 4d) both validate our BioID results that implicate actinin as an interaction

14 hub for RNA-binding proteins, and establish that IGF2BP2 directly binds actinin through specific

15 structural domains.

16 IGF2BP2 localizes ETC transcripts to actinin, which promotes ETC function and

17 cardiomyocyte contraction

18 IGF2BP2 has been shown to bind ETC transcripts in glioblastoma stem cell lines41, and this

19 observation provided the rationale to test whether IGF2BP2 could also be responsible for

20 actinin-localization of ETC transcripts. We employed a genetic knockdown approach using an

21 shRNA that decreased IGF2BP2 protein levels in iPSC-CMs (Fig. 5a), and performed RIP-

22 qPCR in IGF2BP2-depleted iPSC-CMs to quantify actinin-localized ETC transcripts including

23 NDUFA1, NDUFA8, NDUFA11, and COX6B1. IGF2BP2 knockdown decreased these actinin-

24 localized transcripts (Fig. 5b) but not total cellular transcript levels (Fig. 5c)—clarifying that

25 IGF2BP2 is necessary for actinin proximity localization but not stability. Because not all

26 IGF2BP2 is in proximity to actinin (Fig. 4e), and IGF2BP2 may have other functions, we

10 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 sequentially precipitated transcripts by IGF2BP2 antibody first to collect all IGF2BP2-bound

2 transcripts, followed by streptavidin affinity to study the subset of IGF2BP2-bound transcripts

3 that are in actinin proximity (extended data Fig. 5a). We confirmed ETC transcript enrichment at

4 actinin-localized IGF2BP2 (extended data Fig. 5a).

5 Because ETC transcripts were actinin-localized by IGF2BP2, and these transcripts were

6 polysome-enriched, we hypothesized that IGF2BP2 genetic knockdown could impair ETC

7 transcript translation and ETC function. To test this, we measured protein levels of subunits

8 from ETC complexes I-V as well as a glycolytic control, GAPDH. IGF2BP2 knockdown resulted

9 in diminished protein expression across all ETC complexes, but not GAPDH (Fig. 5d).

10 Functionally, IGF2BP2 depletion resulted in reduced mitochondrial content (Fig. 5e) and oxygen

11 consumption rates (Fig. 5f, 5g), which would be consistent with reduced ETC translation.

12 Because cardiomyocytes utilize oxidative metabolism for energy production42, we hypothesized

13 that IGF2BP2 knockdown could impair iPSC-CM functions especially ATP-consuming pathways

14 such as sarcomere contraction. To test contractile function, we knocked-down IGF2BP2 using

15 an shRNA and produced cardiac microtissues (Fig. 5h). In accord with diminished oxidative

16 metabolism, and the energy-dependence of contraction, IGF2BP2 knockdown resulted in

17 decreased twitch contraction (Fig. 5i). To confirm that diminished contraction was not secondary

18 to translational changes in contractility proteins, we quantified beta myosin heavy chain (β-

19 MHC) and troponin I (TnI) levels. Levels of both contractile proteins were unchanged (Fig. 5j).

20 We conclude that IGF2BP2 transports ETC transcripts to the actin cytoskeleton through direct

21 physical interactions with actinin, which supports translation of ETC transcripts, oxidative

22 phosphorylation and contractile function. We propose that IGF2BP2, in partnership with

23 additional actinin-localized factors that regulate RNA-binding, translation and metabolism, form

24 a subcellular spatial hub that links the actin cytoskeleton to metabolic regulation (Fig. 5k).

25

26 Discussion

11 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 The actinins are multipurpose actin cross-linkers that execute their tasks through PPIs with a

2 myriad of partners with diverse biophysical properties and kinetics including the sarcomere,

3 adhesion sites, and transmembrane receptors. Robust methods are necessary to understand

4 the identities, organization and functions of these distinct actin cytoskeletal PPI hubs. Here, we

5 examined the actinin interaction network using proximity-dependent biotinylation by BioID. The

6 vast number of actinin interactions identified in our study was made possible by the unique

7 capabilities of BioID including the ability to detect both direct and vicinal interactions, static and

8 dynamic interactions and interactions in diverse microenvironments such as with membrane-

9 associated partners. In contrast, many actinin PPI studies have utilized yeast two-hybrid

10 approaches that can be challenging with proteins that dimerize and can self-activate yeast

11 reporters43. The robustness of our interaction study was achievable through fusion of BirA* into

12 the endogenous actinin locus made possible with CRISPR/Cas9, which led to Actinin-BirA*

13 expression levels and localization that mimicked unaltered actinin. By targeting the endogenous

14 genetic locus, our approach could also be extended to studies of particularly large proteins

15 including other spectrin family proteins, or to other cell lineages differentiated from iPSCs to

16 understand cell type-specific PPIs.

17 A major objective of this study was to determine the molecular composition of the Z-disk

18 through sarcomere assembly. Our current understanding of Z-disk proteins is derived from

19 stitching together smaller PPI studies focused usually on a single interaction, but has not been

20 systematically characterized especially through a dynamic process such as sarcomere

21 assembly. Here, we combined BioID with a two-stage sarcomere assembly model (i.e. Z-body

22 and Z-disk) and confirmed previous Z-disk proteins including TTN10, TCAP27, LDB344, MYOZ227

23 and CSRP333. CSRP3 was the most-enriched actinin proximity partner with sarcomere

24 assembly, which suggests that CSRP3 may regulate Z-disk organization. While this will require

25 additional studies, it is consistent with observations made by others in mice that CSRP3 genetic

26 disruption impairs Z-disk architecture and results in heart failure45. In addition to sarcomere

12 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 components, we observed sarcomere assembly-dependent interaction partners in functional

2 modules of glycolysis, translation, unfolded protein response, NAD/NADH binding and

3 localization to mitochondrial membranes. Muscle actinin isoforms have been previously found to

4 interact with enzymes related to glucose metabolism including glycogen phosphorylase46,

5 aldolase47 and fructose-bisphosphatase47. Our study confirms actinin-proximity with glycogen

6 phosphorylase (PYGM and PYGB), but also provides a list of additional glycolytic interaction

7 partners including clarifying those that are sarcomere assembly-dependent such as the rate-

8 limiting glycolytic enzyme phosphofructokinase (PFKM). Actinin proximity of PFKM may regulate

9 glucose metabolism through post-translational mechanisms that remain to be explored. In the

10 future, our study could be extended to employ more dynamic approaches such as with the

11 recently-described TurboID enzyme48, which exhibits vastly-improved biotinylation kinetics

12 (~15mins) and labeling efficiencies compared to BirA*.

13 Our BioID study yielded unexpected actinin functions in spatial regulation of RNA

14 localization and translation. In zebrafish, 71% of transcripts are localized in spatially-distinct

15 patterns49, which provides spatial and temporal regulation of gene expression such that local

16 stimuli can regulate translation on-site50. We find that actinin serves as a PPI hub for RNA-

17 binding proteins and translational machinery, which we exploited to identify actinin-localized

18 transcripts that function in the ETC. Our study provides potential new mechanisms for the

19 previous observation that intermyofibrillar mitochondria both reside adjacent to the Z-disk51, and

20 have higher rates of oxidative phosphorylation52. We find that ETC transcript localization to

21 actinin is dependent on interactions with IGF2BP2, an RNA-binding protein associated with type

22 2 diabetes by genome-wide association studies37. In our study, IGF2BP2 knockdown using a 3-

23 dimensional cardiac microtissue model resulted in diminished contractile function in association

24 with reduced ETC translation and oxygen consumption rates. This mechanism may inform why

25 IGF2BP2 knock-out mice also have impaired fatty acid oxidation in skeletal muscle where

26 actinin is also highly expressed53. Finally, our combined proximity proteomic and RNA study

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 provides unexplored areas for investigation for the research community to continue to explore

2 and refine actinin functions.

3

4 Acknowledgements

5 This study could not have been completed without the assistance of Bo Reese (UConn Center

6 for Genome Innovation) for RNA sequencing and RIP-seq, and Anthony Carcio (The Jackson

7 Laboratory for Genomic Medicine) for flow cytometry. TMT proteomics and quantitative

8 analyses was supported by Sanjukta Thakurta from the Thermo Fisher Center for Multiplexed

9 Proteomics at Harvard Medical School. Artwork was created using BioRender. Funding for this

10 study was obtained from the National Institutes of Health (J.T.H., HL125807, HL142787, and

11 EB028898), American Heart Association (F.A.L., PRE35110005, and A.M.P., PRE34381021)

12 and institutional start-up funds (UConn Health).

13

14 Author Contributions

15 Investigation and Validation, F.A.L., A.M.P., K.T., N.L., and J.T.H.; Cells and/or reagent

16 generation, F.A.L., A.M.P., K.T., N.L., R.C., R.R., E.M., Y.S.C., and J.T.H.; Formal Analysis,

17 F.A.L., A.M.P., and J.T.H.; Funding Acquisition, F.A.L., A.M.P., and J.T.H.; Conceptualization

18 and Supervision, J.T.H; Writing, F.A.L., A.M.P., and J.T.H.

19

20 Figure legends:

21 Figure 1. Actinin proximity interaction networks using BioID. a) Overview of actinin

22 localization in cardiomyocytes including sarcomere Z-disk, focal adhesion and cortical actin

23 cytoskeleton. b) Overview of the BioID method using Actinin-BirA* fusion to study actinin

24 proximal protein networks. c) iPSC-CM lysates were probed with anti-actinin and anti-GAPDH to

25 identify Actinin-BirA* fusion (~130kD) and endogenous actinin (~100kD). d) Confocal

26 micrograph of Actinin-BirA* iPSC-CMs decorated with antibodies to actinin (red), streptavidin-

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1 AF488 (green) and DAPI DNA co-stain (blue) (scale bars, main image-10µm, inset-5µm). e) To

2 validate localization, iPSC-CM lysates were immunoprecipitated with an anti-HA antibody, and

3 probed with antibodies to known sarcomere components at Z-disk (anti-TCAP) and M-line (anti-

4 myomesin), as well as anti-HA and anti-actinin controls. f) Overview of BioID experimental

5 methods. g) iPSC-CM lysates were probed with streptavidin-HRP to examine Actinin-BirA*-

6 biotinylated proteins. h) TMT quantitative data illustrated using hierarchical clustering and

7 heatmap confirms low inter-replicate variability and 285 Actinin-BirA*-enriched proteins. i-n)

8 Interaction maps of TMT results based on STRING and GO terms – sarcomere, focal adhesion,

9 cell junction, metabolism, mitochondrial membrane, and RNA-binding. ACTN2 was shaded red

10 to recognize established network interactions (i, j).

11 Figure 2. Sarcomere assembly-dependent actinin proximity interaction networks. a)

12 Confocal micrograph of cTnT-KO Actinin-BirA* iPSC-CMs decorated with antibodies to actinin

13 (red), streptavidin-AF488 (green), and DAPI DNA co-stain (blue) (scale bars, main image-10µm,

14 inset-5µm). In contrast to the Z-disks observed in cTnT-WT iPSC-CMs (fig. 1d), cTnT-KO iPS-

15 CMs have punctate Z-bodies. b) TMT quantitative data illustrated using hierarchical clustering

16 and heatmap for two-stage sarcomere assembly reveal 80 sarcomere assembly-dependent

17 actinin proximity partners (>30% increase in intensity in cTnT-WT compared to cTnT-KO

18 samples). c) List of actinin proximity partners organized by fold-change enrichment with

19 sarcomere assembly. d) STRING interaction map of sarcomere assembly-dependent TMT

20 results analyzed by cluster analysis reveals five clusters labeled by GO enrichment terms.

21 Figure 3. Identifying actinin-localized RNA transcripts using RIP-seq. a) Overview of

22 modified RIP-seq protocol to study actinin proximity RNA transcripts by streptavidin affinity

23 purification of RNA-binding proteins followed by RNA isolation and sequencing. b) PCA plot

24 shows clustering of Actinin-BirA* RIP-seq samples relative to controls (n=4). c) Volcano plot of

25 RIP-seq data shaded red for 945 actinin-enriched transcripts (FDR <0.05, L2FC ≥0.5) and blue

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1 for 1113 actinin-depleted transcripts (FDR <0.05, L2FC ≤-0.5). Differentially-enriched transcripts

2 were analyzed by GO and enrichment terms are listed for actinin-enriched or actinin-depleted

3 samples. d) ETC gene components spanning respiratory chain complex I-V proteins are actinin

4 proximity-enriched on average. Each unshaded circle represents a single ETC gene. e)

5 Glycolysis gene components are not actinin proximity-enriched on average. Each unshaded

6 circle represents a single glycolysis gene. f) Sarcomere gene components divided by

7 localization to the thick or thin filament are not actinin proximity-enriched or are depleted on

8 average, respectively. Each unshaded circle represents a single sarcomere gene. g)

9 Representative confocal micrograph of iPSC-CMs decorated with antibodies to actinin (red),

10 DAPI DNA co-stain (blue), and RNA FISH probes against NDUFA1 (green) and TTN (purple).

11 (scale bars, main image-10µm, inset-5µm). h) Quantification of RNA proximity to actinin. Data

12 are derived from n=10 cells. i) Representative polysome profile of iPSC-CMs used for

13 Polysome-seq studies. j) PCA plot shows clustering of polysome-enriched transcripts compared

14 to controls. k) Venn diagram illustrating unique and overlapping transcripts between Actinin-

15 BirA* RIP-seq and Polysome-seq (FDR <0.05, -0.5 ≤ L2FC ≥ 0.5 for both). L) Overlay of Actinin-

16 BirA* RIP-seq and Polysome-seq results to identify actively translating genes (FDR <0.05, L2FC

17 ≥0.5 by Polysome-seq) that are actinin proximity-enriched (FDR <0.05, L2FC ≥0.5 by RIP-seq),

18 or actinin proximity-depleted (FDR <0.05, L2FC ≤-0.5 by RIP-seq) and the resulting GO terms

19 for each gene set. Data are mean ± SEM; significance assessed by one-way ANOVA using

20 Holm-Sidak correction for multiple comparisons (h).

21 Figure 4. RNA-binding protein IGF2BP2 directly binds actinin. a) Representative

22 immunoblot probed with antibodies to IGF2BP2 in streptavidin affinity-purified lysates. Double

23 band represents two IGF2BP2 splice products. b) Representative immunoblot probed with

24 antibodies to IGF2BP2 in anti-HA immunoprecipitated lysates. Double band represents two

25 IGF2BP2 splice products. c) Mammalian-two-hybrid (M2H) study conducted in 293T cells to fine

26 map the direct interaction between actinin and IGF2BP2. IGF2BP2 was divided into RRM

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 (residues 1-160) and KH (residues 161-600) domains to fine-map the interaction. All data are

2 n=3. The statistical significance was calculated by a paired two-tailed t-test d) M2H results to

3 fine map the direct interaction between the KH domain of IGF2BP2 and Actinin. Actinin was

4 divided into AB (residues 1-275), rod (residues 276-750) and CaM domains (residues 751-895).

5 All data are n=3. e) Representative confocal micrograph demonstrating partial co-localization of

6 actinin (anti-actinin; red) with IGF2BP2 (anti-IGF2BP2; green) with DAPI DNA (blue) co-stain in

7 iPSC-CMs. (scale bars, main image-10µm, inset-5µm). Data are mean ± SEM; significance

8 assessed by one-way ANOVA using Holm-Sidak correction for multiple comparisons (c, d) and

9 defined by P ≤ 0.0001 (****).

10 Figure 5. IGF2BP2 localizes ETC transcripts to actinin, and promotes ETC translation and

11 cardiac microtissue contraction. a) Representative immunoblot of lysates from iPSC-CMs

12 treated with shRNA targeting IGF2BP2 or scramble control probed with antibodies to IGF2BP2

13 and GAPDH. Double band represents two IGF2BP2 splice products. b) Streptavidin affinity

14 purification of transcripts bound to actinin proximity-enriched targets after IGF2BP2 knockdown,

15 followed by qPCR of ETC transcripts NDUFA1, NDUFA8, NDUFA11 and COX6B1. c) QPCR

16 analysis of global transcript levels of NDUFA1, NDUFA8, NDUFA11 and COX6B1 after

17 IGF2BP2 knockdown. d) Representative immunoblot of lysates from iPSC-CMs after IGF2BP2

18 knockdown, and probed with antibodies to ETC complex I-V subunits (anti-ATP5A, UQCRC,

19 MTCO1, SDHB and NDUFB8), IGF2BP2 and GAPDH control. e) FACs analysis of iPSC-CMs

20 stained with MitoTracker after IGF2BP2 knockdown (N>5000 events per replicate). f) Oxygen

21 consumption rates normalized to protein levels after IGF2BP2 knockdown in iPSC-CMs, and (g)

22 specific OCR parameter quantification. h) Overview of cardiac microtissue platform and twitch

23 force analysis. ECM-extracellular matrix slurry. Fibroblasts-normal human ventricular fibroblasts.

24 (scale bar-150µm). i) Cardiac microtissues exhibit reduced twitch force after IGF2BP2

25 knockdown. Data are n≥10 microtissues. j) Representative immunoblot of lysates from iPSC-

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 CMs probed with antibodies to myosin heavy chain 7 (b-MHC) and troponin I (TnI) proteins

2 show no change in expression despite IGF2BP2 knockdown. Anti-GAPDH used as additional

3 control. k) Proposed model of an actinin-based PPI hub that promotes localized oxidative

4 phosphorylation (OXPHOS) through proximity interactions with RNA binding proteins (RBP)

5 including IGF2BP2, translational machinery, unfolded protein response (UPR), and

6 mitochondria. Data are n≥3; mean ± SEM; significance assessed by Student’s t-test (a-e, g, i, j)

7 and defined by P ≤ 0.05 (*), P ≤ 0.01 (**).

8

9 Materials and Methods

10 iPSC Culture and iPSC-CM Differentiation

11 All stem cell experiments were done in PGP1 iPSCs (Coriell #GM23338), which have been

12 extensively characterized including whole genome sequencing and karyotyping as part of the

13 ENCODE project (ENCBS368AAA). iPSCs were maintained on Matrigel-coated tissue culture

14 plates (Corning 354230) in mTeSR1 (STEMCELL Technologies 85875). iPSCs were passaged

15 at 80-90% confluency utilizing Accutase (BD 561527) and 10 μM ROCK inhibitor Y-27632

16 (Tocris 1254). iPSC-CMs were directly differentiated by sequential modulation of Wnt/β-catenin

17 signaling, as previously described21, 54. On Day 13 of differentiation, iPSC-CMs were purified by

18 metabolic enrichment using glucose-free DMEM (Gibco 11966025) supplemented with 4 mM

19 lactate (Sigma 71718) for 24-48 hours18. Following selection, iPSC-CMs were trypsinized (Gibco

20 25200056) and re-plated onto fibronectin-coated tissue culture plates (Gibco 33016015). IPSC-

21 CMs were maintained in RPMI-B27. For proximity-labeling experiments, control and Actinin-

22 BirA* iPSC-CMs were maintained in DMEM (Gibco 11965092) supplemented with homemade

23 biotin-free B27 following metabolic enrichment55, 56. For BioID studies, control and Actinin-BirA*

24 iPSC-CMs were treated with 50 μM biotin for 24 hours followed by a 2-hour washout phase in

25 biotin-free media before collection. All iPSC-CM analyses were performed on Day 25-30 unless

26 noted otherwise.

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1 Plasmid Cloning

2 The homologous recombination (HR) targeting vector (System Biosciences HR220PA-1) was

3 modified prior to being used in genome-editing experiments. A gene fragment for BirA*

4 (R118G)-HA tag was obtained (IDT) and cloned into the parent vector via Gibson assembly

5 (NEB E2621). ACTN2 ~800bp 5’ and 3’ HR arm gene fragments (IDT) were then cloned into the

6 appropriate cloning sites (sequences in Table S5). All HR vector propagation steps were

7 performed in DH5α E.coli (NEB C2987). For hairpin (shRNA) experiments, the pLKO.1 puro

8 lentiviral plasmids containing either shScramble (Addgene 1864) or shIGF2BP2 (sequence in

9 Table S5) were assembled using T4 DNA ligase (NEB M0202S) and propagated in NEB Stable

10 E.coli (NEB C3040).

11 CRISPR/Cas9 studies

12 Genome editing was performed utilizing a CRISPR/Cas9 protocol adapted from previous

13 studies20, 21. 8x106 iPSCs were electroporated with 20 μg pCas9-GFP (Addgene 44719), 20 μg

14 of the appropriate hU6-driven sgRNA (designed using crispr.mit.edu), and 20 μg of the HR

15 targeting vector to generate ACTN2-BirA* knock-in iPSCs. Electroporated cells were transferred

16 to a Matrigel-coated 100 mm dish containing mTeSR1 and 10 μM Y-27632. The following day,

17 selection was started with 50 μg/mL Hygromycin B (Invitrogen 10687010) to isolate single iPSC

18 clones, which were then manually picked, expanded, and screened via Sanger sequencing.

19 Isogenic knockout of TNNT2 was performed similarly, but in the absence of an HR vector and

20 with GFP-based FACS enrichment in place of antibiotic selection.

21 Immunoprecipitation Experiments

22 Streptavidin immunoprecipitation

23 After biotin washout, control and Actinin-BirA* iPSC-CMs were rinsed three times with room

24 temperature PBS. Cells were lysed with lysis buffer (50 mM Tris-Cl pH7.4, 500 mM NaCl, 0.2%

25 SDS, 1x protease inhibitor, 1mM DTT, ddH2O). Lysates were sonicated (Branson 250) two

26 times on ice with 2 minutes between each cycle (30 pulses, 30% duty cycle, output level3).

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1 Samples were centrifuged to pellet cell debris and cleared lysate were added to magnetic

2 Dynabeads MyOne Streptavidin C1 beads (Invitrogen 65001), which were prewashed with PBS

3 and lysis buffer. Immunoprecipitation was performed on a rotator overnight at 4°C. The following

4 day, the beads were resuspended in wash buffer 1 (2% SDS, ddH2O), rotated for 8 minutes at

5 room temperature, and then re-collected using a magnetic separation stand. The same steps

6 were then repeated with wash buffer 2 (0.1% deoxycholic acid, 1% triton X-100, 1mM EDTA,

7 500 mM NaCl, 50 mM HEPES pH7.5, ddH2O) and wash buffer 3 (0.5% deoxycholic acid, 0.5%

8 NP-40, 1 mM EDTA, 250 mM LiCl, 10 mM Tris-Cl pH7.4, ddH2O). Finally, the washed beads

9 were resuspended in PBS, snap frozen, and sent for quantitative proteomics.

10 HA immunoprecipitation

11 Control and Actinin-BirA* iPSC-CMs were collected at Day 25 and HA-tag IP was conducted

12 using reagents supplied in HA-tag IP/Co-IP Application set (Thermo Scientific 26180Y). The

13 subsequent protein immunoblots were conducted as outlined in the immunoblot section.

14 RNA Immunoprecipitation (RIP)

15 After biotin washout, control and Actinin-BirA* iPSC-CMs were rinsed three times with room

16 temperature PBS. Cells were lysed with ice-cold RIP buffer (150 mM KCl, 25 mM Tris-Cl pH7.4,

17 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 1x protease inhibitor, 100 U/ml RNase inhibitor, RNase-

18 free water). Cell debris was pelleted and cleared lysate was added to magnetic Dynabeads

19 MyOne Streptavidin C1 beads (Invitrogen 65001), which were prewashed with PBS and RIP

20 buffer. Samples and beads were incubated on a rotator overnight at 4°C. The following day, the

21 beads were washed three times with ice-cold RIP buffer – all steps were performed in a 4°C

22 room. Samples were then incubated with Proteinase K at 55°C for 30 minutes. Beads were

23 resuspended in TRIzol (Invitrogen 15596018), RNA extraction was performed immediately

24 followed by RNA-sequencing.

25 Tandem Immunoprecipitation

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1 After biotin washout, control and Actinin-BirA* iPSC-CMs were rinsed three times with room

2 temperature PBS and then lysed in RIP buffer. Cleared lysates were incubated with Dynabeads

3 Protein G (Invitrogen 10003D) pre-bound to either IgG antibody (MBL PM035) or IGF2BP2

4 antibody (MBL RN008P). Samples and beads were incubated on a rotator overnight at 4°C. The

5 following day, the Protein G beads were washed three times with RIP buffer and then incubated

6 for 12 hours with rotation at 4°C in RIP buffer containing IMP2 peptide (Genescript). The

7 supernatant was then mixed with magnetic Dynabeads MyOne Streptavidin C1 beads

8 (Invitrogen 65001), which were prewashed with PBS and RIP buffer. The following day, the

9 streptavidin beads were three times washed with ice-cold RIP buffer – all steps were performed

10 in a 4°C room. Samples were then incubated with Proteinase K at 55°C for 30 minutes. Beads

11 were resuspended in TRIzol, RNA extraction was performed immediately followed by cDNA

12 synthesis (outlined in Quantitative PCR section).

13 Quantitative proteomics

14 Bead digestion

15 After conducting streptavidin immunoprecipitation, streptavidin beads were snap frozen and

16 sent to the Thermo Fisher Center for Multiplexed Proteomics (TCMP) at Harvard Medical

17 School. There, beads were washed three times with 50 mM Tris buffer (pH 8.0) to remove any

18 residual contents and resuspended in 1M Urea, 50 mM Tris (pH 8.0). An initial digest was

19 performed for 1 hour at 37°C with Trypsin (5ng/ul), followed by brief centrifugation and

20 supernatant collection. Beads were washed three time with 1M Urea, 50 mM Tris (pH 8.0),

21 washed fractions were pooled, and digest was continued overnight at room temperature. The

22 next morning, digested peptides were reduced with 5 mM TCEP, alkylation with 10 mM

23 Iodoacetamide, quenching alkylation with 5 mM DTT, and final quenching with TFA. Stage-tip

24 desalting on acidified digested peptides was conducted as previously described57. Peptide were

25 eluted with 80% acetonitrile, 0.1% TFA and dried with speedvac. Dried desalted peptide

26 samples were reconstituted with 200 mM EPPS buffer (pH 8.0) and labelled with first 9 of a 10-

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1 plex tandem mass tag (TMT) reagent. 9-plex labeling reactions were conducted for 1 hour at

2 room temperature. Modification of tyrosine residues with TMT was reversed with the addition of

3 5% hydroxyl amine for 15 minutes and the reaction was quenched with 0.5% TFA. Next,

4 samples were combined, desalted over stage-tip, eluted into Autosampler Inserts, dried in a

5 speedvac, and reconstituted in 5% acetonitrile-5% TFA for next steps.

6 Liquid chromatorgraphy-MS3 spectrometry (LC-MS/MS)

7 Labelled samples were analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher

8 Scientific) with a Thermo Easy-nLC 1200 for online sample handling and peptide separations58.

9 Peptides were separated using a 180 min linear gradient from 5% to 42% buffer B (90% ACN +

10 0.1% formic acid) equilibrated with buffer A (5% ACN + 0.1% formic acid) at a flow rate of 500

11 nL/min across the column. MS1 data was collected in Orbitrap using a resolution of 120,000

12 with a scan range of 350-1350 m/z with a maximum injection time of 50 ms. MS2 analysis

13 consisted of CID (quadrupole isolation set at 0.7 Da and ion trap analysis, AGC 9 × 103,

14 Collision Energy 35%, maximum injection time 80 ms). The top ten precursors from each MS2

15 scan were selected for MS3 analysis (synchronous precursor selection), in which precursors

16 were fragmented by HCD prior to Orbitrap analysis (Collision Energy 55%, max. AGC 1 × 105,

17 maximum injection time 120 ms, resolution 50,000 and isolation window set to 1.2 – 0.8).

18 LC-MS3 data analysis

19 Uniprot Human database was used to search forward and reverse sequences of MS/MS

20 spectra. The follow criteria were used: tryptic with two missed cleavages, a precursor mass

21 tolerance of 50 ppm, fragment ion mass tolerance of 1.0 Da, static alkylation of cysteine

22 (57.02146 Da), static TMT labeling of lysine residues and N-termini of peptides (229.162932

23 Da), and variable oxidation of methionine (15.99491 Da). Poor quality MS3 spectra were

24 excluded from quantification (<200 summed signal-to-noise across 10 channels and <0.5

25 precursor isolation specificity).

26 Bioinformatics analysis

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 For analysis, raw intensity values underwent +1 correction prior to log transformation and

2 subsequent enrichment analysis. Search Tool for the Retrieval of Interacting Genes/Proteins

3 (STRING v. 11.0) was utilized for generation of interaction scores and Gene ontology (GO) term

4 enrichment and KEGG Pathway analysis provided in supplemental tables. Cytoscape (v.3.7.2)

5 was utilized to generate interaction maps with an organic layout and the utilization of the

6 CLUSTER and BINGO features for group clustering and appropriate GO term identification for

7 each cluster. Quantitative proteomics raw data deposited to the ProteomeXchange Consortium

8 identifier PXD018040.

9 Quantitative PCR, RNA sequencing, and analysis

10 cDNA was synthesized using Superscript III First-Strand synthesis (Invitrogen 18080-400).

11 Gene-specific PCR primers were designed or identified from the literature and transcripts were

12 quantified using Fast SYBR Green (Applied Biosystems 4385612) on a ViiA7 Real-Time PCR

13 system (Applied Biosystems).

14 RNA Samples were sent for sequencing at the University of Connecticut Institute for

15 Systems Genomics. RNA sequencing libraries were generated using Illumina TruSeq Stranded

16 Total RNA library preparation (Ribo-Zero depletion for rRNA was utilized for RIP-seq). Illumina

17 NextSeq 500/550 sequencing was conducted with the v2.5 300 cycle reagent kit 9 (High

18 Output). Estimated total single end reads per sample = 30-35M 150bp PE reads. Samples

19 sequences were aligned with STAR to the hg38 human genome. In order to look at differential

20 expression, DESeq2 (Bioconductor) was utilized. Gene Set Enrichment Analysis (GSEA) was

21 utilized to determine GO terms for data sets. Top 10 GO terms are presented. Furthermore, for

22 ETC complexes, glycolysis, and sarcomere gene sets – GSEA gene sets were utilized and

23 those with an average ≥5 TPM value were included in the data set. Genome sequencing

24 datasets are deposited at GEO under accession numbers: GSE144805 and GSE144806.

25 Protein Immunoblotting

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 Samples of interest were lysed in RIPA buffer (Cell Signaling 9806) containing protease inhibitor

2 cocktail (Roche 11836170001), 1 mM PMSF, and phosphatase inhibitor (Pierce A32957),

3 unless noted otherwise. Protein lysate concentrations were normalized using Pierce BCA

4 (Thermo 23225), and then reduced and denatured in sample buffer (Thermo 39000). Lysates

5 were separated on Bio-Rad 4–20% Mini-PROTEAN TGX precast gels, transferred via Bio-Rad

6 Trans-Blot Turbo onto PVDF membranes (Bio-Rad 1704272), blocked in TBS-T (TBS with 0.1%

7 Tween-20) containing 5% BSA, and probed overnight at 4°C with primary antibody. The next

8 day, blots were washed in TBS-T and probed with HRP-linked secondary antibody (Cell

9 Signaling 7076; 7074; Streptavidin-HRP 3999S) for 1 hour. Signal detection was performed

10 using ECL substrate (Thermo 34577; 34095) and a Bio-Rad ChemiDoc MP imaging system.

11 Blot images were digitally processed and analyzed in ImageJ. The primary antibodies used

12 were as follows: anti-actinin (Cell Signaling 6487), anti-OxPhos (Invitrogen 458199) anti-HA

13 (Cell Signaling 14793), anti-IGF2BP2 (MBL RN008P), anti-PBCBP1 (Abcam 74793), anti-

14 PCBP2 (MBL RN250P), anti-SERBP1 (Abcam 55993), anti-TCAP (BD 612328), and anti-

15 MYOM (mMaC myomesin B4 was deposited to the DSHB by Perriard, J.-C.).

16 Immunofluorescence and RNA FISH

17 For standard immunofluorescence images, cells were fixed on coverslips (Fisherbrand 12-545-

18 100) in PBS containing 4% paraformaldehyde (EMS 50-980-487) and then permeabilized and

19 blocked in PBS-T (PBS containing 0.1% Triton-X) with 1% BSA (Fisher BP1605100). Cells were

20 probed with primary antibody in PBS-T with BSA overnight at 4°C. The following primary

21 antibodies were used:, anti-Actinin (Sigma A7811) and anti-IGF2BP2 (Bethyl A303-316A). The

22 next day coverslips were washed in PBS-T, incubated for 1 hour at room temperature with 1

23 μg/mL DAPI (Invitrogen D1306) and appropriate secondary antibody (Invitrogen A-11005 or A-

24 11008) or streptavidin-488 (Invitrogen S11223), washed, and mounted onto slides (Corning

25 2948) in ProLong Diamond mountant (Invitrogen P36965). An Andor Dragonfly 500 confocal

26 microscope system using a Zyla sCMOS camera and a Leica DMi8 63x oil immersion lens was

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 used for imaging slides. Andor Fusion software was used to acquire images, which were

2 converted to OME-TIFF format using Bitplane Imaris and analyzed in ImageJ.

3 ViewRNA direct fluorescence RNA in situ hybridization was conducted using the

4 manufacturer’s protocol (Invitrogen 19887). The following human probes were utilized: NDUFA1

5 (Invitrogen VA6-3172657), NDUFA8 (Invitrogen VA1-3003564), GPX4 (Invitrogen VA1-

6 3000349), and TTN (Invitrogen VA6-3173894). Final step of coverslip mounting and image

7 acquisition are described above. For distance quantification, Imaris software was utilized.

8 Images first underwent background subtraction to minimize background signal and allow for

9 clear puncta visualization. The distance transformation tool on Imaris was used to quantify

10 distance (in microns) of RNA puncta and actinin structures.

11 Mammalian-Two Hybrid

12 Actinin (full-length and domains), IGF2BP2 (full-length and domains), PCBP1, PBCBP2, and

13 SERBP1 were amplified from human cDNA and cloned into the appropriate vectors (pACT or

14 pBIND) provided by the CheckMate Mammalian Two-Hybrid kit (Promega E2440). The

15 appropriate combination of edited pACT and pBIND vectors, in addition to the kit-provided

16 pGluc5 vector, were transfected using PEI into HEK 293T cells (ATCC CRL-3216), as outlined

17 in the manufacturer’s protocol. In addition, the kit-provided positive and negative controls were

18 transfected simultaneously for all experiments to verify proper signal production. Luciferase and

19 Renilla activity were measured using manufacturer’s protocol (Promega E1910) utilizing

20 BioTek’s Synergy 2 multi-mode microplate reader.

21 Lentivirus Production

22 HEK 293T cells (ATCC CRL-3216) were seeded onto 150 mm plates in 20 mL DMEM (Gibco

23 11965092) supplemented with 10% FBS (Gemini 100-106), GlutaMAX (Gibco 35050061), and 1

24 mM sodium pyruvate (Gibco 11360070). HEK 293T cells were transfected with 18ug of lentiviral

25 transfer vector, 12 μg psPAX2 (Addgene 12260), and 6 μg pCMV-VSV-G (Addgene 8454).

26 Media was replenished the following day and virus-containing media was harvested at 48, 72,

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 and 96 hours post-transfection, followed by concentration using PEG-6000 as previously

2 described59. For titer determination - iPSCs were transduced with a serial dilution of lentivirus,

3 followed by treating with appropriate antibiotic (1µg/ml puromycin), and counting resistant

4 colony-forming units.

5 Polysome profiling

6 IPSC-CMs at day 25 were treated with 0.1 mg/ml cycloheximide at 37 °C for 10 minutes. Cells

7 were washed with 0.1 mg/ml cycloheximide in PBS and lysed in polysome lysis buffer (10 mM

8 HEPES ph7.3, 150 mM KCl, 10 mM MgCl2, 1 mM DTT, 100ug/ml cycloheximide, 2% NP-40,

9 protease inhibitor mini tablet, 6U/ml RNase inhibitor). Samples were kept on ice for 5 minutes

10 during lysis and then centrifuged at 13000g for 10 minutes at 4 °C to remove debris. The

11 BioComp Gradient Station ip Model 153 was used to prepare sucrose gradients. 15% and 50%

12 sucrose solutions (sucrose diluted in 10 mM HEPES pH 7.3, 150 mM KCl, 10 mM MgCl2, 100

13 ug/ml cycloheximide, 1mM DTT) were utilized. Supernatants from lysed cells were added to

14 sucrose gradients and centrifuged for 150 minutes at 4°C at 120000g (SW41 rotor, Beckman

15 Coulter Optima XPN-100 Ultracentrifuge). BiComp Gradient Station and TRIAX software were

16 utilized for separation of sucrose gradient fractions. Fractions were collected, resuspended in

17 TRIzol and RNA extraction was conducted followed by RNA-Sequencing

18 Individual Gene Knockdown

19 Lentiviral-based hairpins were used to individually knockdown genes directly in iPSC-CMs.

20 iPSC-CMs were transduced with lentivirus expressing hairpin at a MOI of 2 in RPMI-B27. Media

21 was replenished the following day and cells were analyzed 7-10 days post transduction.

22 Flow Cytometry Analysis

23 BD Biosciences FACSymphony A5 was utilized to perform all flow cytometry experiments. Cells

24 were stained in suspension for 30 minutes at 37°C with 10 μM Hoechst 33342 (Thermo 62249),

25 200 nM MitoTracker Green (Invitrogen M7514), and 500 nM TO-PRO-3 (Invitrogen T3605).

26 Cells were gated based on forward-scatter and side-scatter, followed by gating of TO-PRO-3 to

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 identify live cells. Hoechst was used to gate for single cells, followed by subsequent analysis of

2 mitochondrial content (MitoTracker). Final analysis was performed using FlowJo software.

3 Mito Stress Test

4 30,000 wildtype CMs were plated on Seahorse XFe96 Cell Culture Microplates (Agilent 101085-

5 004) and lentiviral shRNAs (Scramble and IGF2BP2) were added the next day. The cells were

6 cultured for 7 days before performing the Seahorse XF Cell Mito Stress Test (Agilent 103015-

7 100) according to manufacturer’s protocol. The following final concentrations of compounds

8 were injected: oligomycin (1 μM), FCCP (1 μM), and rotenone/antimycin A (0.5 μM).

9 Cardiac microtissue assay

10 Cardiac tissues were generated as previously described20, 21. Briefly, polydimethylsiloxane

11 (PDMS) (Corning Sylgard 184) cantilever devices were molded from SU-8 masters, which were

12 embedded with fluorescent microbeads (Thermo F8820). Singularized iPSC-CMs were mixed

13 with human cardiac fibroblasts and spun into PDMS devices containing a collagen-based ECM.

14 For knockdown experiments, lentiviral shRNAs were added to iPSC-CMs 7 days prior to

15 generation of tissues. Tissue function was measured once tissues compacted and were visibly

16 pulling the cantilevers. For acquisition of functional data, tissues were stimulated at 1 Hz with a

17 C-Pace EP stimulator (IonOptix) with platinum wire electrodes and fluorescence images were

18 taken at 25 Hz with a Andor Dragonfly microscope equipped with enclosed live-cell chamber

19 (Okolabs). Displacement of fluorescent microbeads was tracked using the ImageJ

20 ParticleTracker plug-in and twitch force was calculated. The cantilever spring constants were

21 determined using the elastic modulus of PDMS and the dimensions of the tissue gauge devices

22 previously described60.

23 Statistical Analysis

24 Obtained data were analyzed in Microsoft Excel and graphed using either GraphPad Prism or

25 R. Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons

26 were conducted via Student’s t-test or one-way ANOVA followed by multiple comparisons using

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 Holm-Sidak correction, where appropriate. Statistical significance was defined by p ≤ 0.05 (*), p

2 ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

3 Extended Data

4 Figure 1. BioID to establish actinin proximity partners in iPSC-CMs. a) Overview of gene-

5 targeting strategy to generate an in-frame knock-in of BirA*-HA at the ACTN2 locus in iPSCs.

6 CRISPR/Cas9 and homology-directed repair from a donor vector containing BirA*-HA and

7 800bp ACTN2 homology arms was utilized. b) Actinin-BirA* cardiac microtissues have similar

8 twitch force compared to controls. Data are n≥10 microtissues. (scale bar-150µm). c)

9 Representative protein blot probed with streptavidin-HRP to optimize biotin labeling after 24

10 hours of biotin supplementation up to 50 µM. d) Complete STRING network map of Actinin

11 proximity proteins. Data are mean ± SEM; significance assessed by Student’s t-test (b) and

12 defined by P > 0.05 (ns).

13 Figure 2. BioID through sarcomere assembly. a) Representative immunoblot of cTnT-KO

14 and cTnT-WT iPSC-CMs lysates probed for cardiac troponin T (cTnT), actinin and GAPDH

15 control. b) Representative protein blot of cTnT-KO and cTnT-WT Actinin-BirA* lysates probed

16 with streptavidin-HRP. c) FACS quantification of MitoTracker fluorescence intensity in cTnT-KO

17 and WT iPSC-CMs (N>5000 events). d) Oxygen consumption rates (OCR) with e) parameter

18 quantification of cTnT-KO and cTnT-WT iPSC-CMs. Data are n≥3; mean ± SEM; significance

19 assessed by Student’s t-test (c, e) and defined by P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001

20 (****).

21 Figure 3. RIP-seq to identify actinin proximity RNA transcripts. a) Heatmap displaying

22 relative expression of ETC Complexes (I-V) genes in Control and Actinin-BirA* RIP-seq

23 samples. b) Heatmap displaying relative expression of glycolysis genes in Control vs. Actinin-

24 BirA* RIP-seq samples. c) Heatmap displaying relative expression of sarcomere thin and thick

25 filament genes in Control vs. Actinin-BirA* RIP-seq samples. d) qPCR validation of candidate

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 RIP-seq hits. Data are n=4. e) RNA FISH of ETC Complex I components NDUFA1 and

2 NDUFA8 that are both in proximity to Actinin but not overlapped (scale bars, main image-10µm,

3 inset-5µm). f) Overlap of Actinin-BirA* RIP-seq with Polysome-seq data demonstrates 273

4 actinin-enriched and translated transcripts. ETC Complex I components are denoted with

5 shaded red circles. g) Overlap of Actinin-BirA* RIP-seq with Polysome-seq data demonstrates

6 87 actinin-depleted and translated transcripts. Sarcomere components are denoted with shaded

7 blue circles. Data are mean ± SEM; significance assessed by Student’s t-test (d) and defined by

8 P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****).

9 Figure 4. RNA-binding proteins interact with actinin. a) Representative immunoblot probed

10 with antibodies to PCPB1, PCPB2 and SERBP1 in streptavidin affinity-purified lysates. b)

11 Representative immunoblot probed with antibodies to PCPB1, PCPB2 and SERBP1 in anti-HA

12 immunoprecipitated lysates. c) M2H results demonstrating that IGF2BP2 but not PCBP1,

13 PCPB2 and SERBP1 interact with full-length actinin. d) Summary overview of RBP and actinin

14 interaction data. e) M2H results demonstrating that all actinin isoforms (ACTN1-4) interact with

15 IGF2BP2. Data are n=3; mean ± SEM; significance assessed by one-way ANOVA using Holm-

16 Sidak correction for multiple comparisons (c, e) and defined by P ≤ 0.0001 (****).

17 Figure 5. IGF2BP2 localizes ETC transcripts to actinin. a) Overview of tandem-IP process

18 (left) and qPCR results of IgG vs. IGF2BP2 IP (right) for candidate ETC transcripts. Data are

19 n=3; mean ± SEM; significance assessed by Student’s t-test (a) and defined by P ≤ 0.05 (*). b)

20 Gating strategy utilized for analysis of mitochondrial content for Main Figure 5e.

21

22 Table 1. TMT summary data for actinin-proximity proteins. a) Composite protein hits. b) Exp. 1

23 raw intensity. c) Exp. 2 raw intensity.

24 Table 2. Actinin-proximity TMT proteins and STRING/GO analyses. a) Protein hits. b) STRING

25 – GO terms. c) STRING – KEGG pathway.

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 Table 3. Sarcomere assembly-dependent TMT proteins and STRING/GO analyses. a) Protein

2 hits. b) STRING – GO terms. c) STRING – KEGG pathway.

3 Table 4. RIP-seq data and GO Terms. a) Gene list. b) GO terms (enriched). c) GO terms

4 (depleted).

5 Table 5. Overlay of RIP-seq and Polysome-seq data and GO terms. a) Combined gene list. b)

6 Gene list for polysome active and RIP enriched. c) GO terms for polysome active and RIP

7 enriched. d) Gene list for polysome active and RIP depleted. e) GO terms for polysome active

8 and RIP depleted.

9 Table 6. CRISPR and primer sequences.

10 Movie 1. Control Actinin-BirA* iPSC-CMs paced at 1Hz. Magnification-5X. Scale bar-150µm.

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 References

2 1. Liem, R.K. Cytoskeletal Integrators: The Spectrin Superfamily. Cold Spring Harb 3 Perspect Biol 8 (2016). 4 2. Ribeiro Ede, A., Jr. et al. The structure and regulation of human muscle alpha-actinin. 5 Cell 159, 1447-1460 (2014). 6 3. Virel, A. & Backman, L. Molecular evolution and structure of alpha-actinin. Mol Biol 7 Evol 21, 1024-1031 (2004). 8 4. Murphy, A.C. & Young, P.W. The actinin family of actin cross-linking proteins - a 9 genetic perspective. Cell Biosci 5, 49 (2015). 10 5. Kunishima, S. et al. ACTN1 mutations cause congenital macrothrombocytopenia. Am J 11 Hum Genet 92, 431-438 (2013). 12 6. Chiu, C. et al. Mutations in alpha-actinin-2 cause hypertrophic cardiomyopathy: a 13 genome-wide analysis. J Am Coll Cardiol 55, 1127-1135 (2010). 14 7. Kaplan, J.M. et al. Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal 15 segmental glomerulosclerosis. Nat Genet 24, 251-256 (2000). 16 8. Chopra, A. et al. Force Generation via beta-Cardiac Myosin, Titin, and alpha-Actinin 17 Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions. Dev Cell 44, 87-96 18 e85 (2018). 19 9. Schultheiss, T. et al. A sarcomeric alpha-actinin truncated at the carboxyl end induces the 20 breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and 21 breakdown of myofibrils in myotubes. Proc Natl Acad Sci U S A 89, 9282-9286 (1992). 22 10. Grison, M., Merkel, U., Kostan, J., Djinovic-Carugo, K. & Rief, M. alpha-Actinin/titin 23 interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk. 24 Proc Natl Acad Sci U S A 114, 1015-1020 (2017). 25 11. Sanger, J.M. & Sanger, J.W. The dynamic Z bands of striated muscle cells. Sci Signal 1, 26 pe37 (2008). 27 12. Lange, S., Ehler, E. & Gautel, M. From A to Z and back? Multicompartment proteins in 28 the sarcomere. Trends Cell Biol 16, 11-18 (2006). 29 13. Djinovic-Carugo, K., Gautel, M., Ylanne, J. & Young, P. The spectrin repeat: a structural 30 platform for cytoskeletal protein assemblies. FEBS Lett 513, 119-123 (2002). 31 14. Foley, K.S. & Young, P.W. The non-muscle functions of actinins: an update. Biochem J 32 459, 1-13 (2014). 33 15. Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein 34 identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196, 801-810 35 (2012). 36 16. Gingras, A.C., Abe, K.T. & Raught, B. Getting to know the neighborhood: using 37 proximity-dependent biotinylation to characterize protein complexes and map organelles. 38 Curr Opin Chem Biol 48, 44-54 (2019). 39 17. Kim, D.I. et al. Probing nuclear pore complex architecture with proximity-dependent 40 biotinylation. Proc Natl Acad Sci U S A 111, E2453-2461 (2014). 41 18. Tohyama, S. et al. Distinct metabolic flow enables large-scale purification of mouse and 42 human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127-137 (2013). 43 19. Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells 44 via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A 109, 45 E1848-1857 (2012).

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 20. Hinson, J.T. et al. HEART DISEASE. Titin mutations in iPS cells define sarcomere 2 insufficiency as a cause of dilated cardiomyopathy. Science 349, 982-986 (2015). 3 21. Cohn, R. et al. A Contraction Stress Model of Hypertrophic Cardiomyopathy due to 4 Sarcomere Mutations. Stem Cell Reports 12, 71-83 (2019). 5 22. Auerbach, D. et al. Different domains of the M-band protein myomesin are involved in 6 myosin binding and M-band targeting. Mol Biol Cell 10, 1297-1308 (1999). 7 23. Gautel, M. & Djinovic-Carugo, K. The sarcomeric cytoskeleton: from molecules to 8 motion. J Exp Biol 219, 135-145 (2016). 9 24. Hayashi, T. et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated 10 cardiomyopathy. J Am Coll Cardiol 44, 2192-2201 (2004). 11 25. Lyon, R.C., Zanella, F., Omens, J.H. & Sheikh, F. Mechanotransduction in cardiac 12 hypertrophy and failure. Circ Res 116, 1462-1476 (2015). 13 26. Szklarczyk, D. et al. STRING v11: protein-protein association networks with increased 14 coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic 15 Acids Res 47, D607-D613 (2019). 16 27. Faulkner, G. et al. FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z- 17 disc of skeletal muscle. J Biol Chem 275, 41234-41242 (2000). 18 28. Crawford, A.W., Michelsen, J.W. & Beckerle, M.C. An interaction between zyxin and 19 alpha-actinin. J Cell Biol 116, 1381-1393 (1992). 20 29. Nieset, J.E. et al. Characterization of the interactions of alpha-catenin with alpha-actinin 21 and beta-catenin/plakoglobin. J Cell Sci 110 ( Pt 8), 1013-1022 (1997). 22 30. Du, A., Sanger, J.M., Linask, K.K. & Sanger, J.W. Myofibrillogenesis in the first 23 cardiomyocytes formed from isolated quail precardiac mesoderm. Dev Biol 257, 382-394 24 (2003). 25 31. Nishii, K. et al. Targeted disruption of the cardiac troponin T gene causes sarcomere 26 disassembly and defects in heartbeat within the early mouse embryo. Dev Biol 322, 65-73 27 (2008). 28 32. Knoll, R. et al. The cardiac mechanical stretch sensor machinery involves a Z disc 29 complex that is defective in a subset of human dilated cardiomyopathy. Cell 111, 943- 30 955 (2002). 31 33. Heineke, J. et al. Attenuation of cardiac remodeling after myocardial infarction by 32 muscle LIM protein-calcineurin signaling at the sarcomeric Z-disc. Proc Natl Acad Sci U 33 S A 102, 1655-1660 (2005). 34 34. Knoll, R. et al. A common MLP (muscle LIM protein) variant is associated with 35 cardiomyopathy. Circ Res 106, 695-704 (2010). 36 35. Tenenbaum, S.A., Carson, C.C., Lager, P.J. & Keene, J.D. Identifying mRNA subsets in 37 messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S 38 A 97, 14085-14090 (2000). 39 36. Pringle, E.S., McCormick, C. & Cheng, Z. Polysome Profiling Analysis of mRNA and 40 Associated Proteins Engaged in Translation. Curr Protoc Mol Biol 125, e79 (2019). 41 37. Diabetes Genetics Initiative of Broad Institute of, H. et al. Genome-wide association 42 analysis identifies loci for type 2 diabetes and triglyceride levels. Science 316, 1331-1336 43 (2007). 44 38. Ryu, M.S., Zhang, D., Protchenko, O., Shakoury-Elizeh, M. & Philpott, C.C. PCBP1 and 45 NCOA4 regulate erythroid iron storage and heme biosynthesis. J Clin Invest 127, 1786- 46 1797 (2017).

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 39. Frey, A.G. et al. Iron chaperones PCBP1 and PCBP2 mediate the metallation of the 2 dinuclear iron enzyme deoxyhypusine hydroxylase. Proc Natl Acad Sci U S A 111, 8031- 3 8036 (2014). 4 40. Muto, A. et al. The mRNA-binding protein Serbp1 as an auxiliary protein associated with 5 mammalian cytoplasmic ribosomes. Cell Biochem Funct 36, 312-322 (2018). 6 41. Janiszewska, M. et al. Imp2 controls oxidative phosphorylation and is crucial for 7 preserving glioblastoma cancer stem cells. Genes Dev 26, 1926-1944 (2012). 8 42. Suga, H. Ventricular energetics. Physiol Rev 70, 247-277 (1990). 9 43. Frey, N. & Olson, E.N. Calsarcin-3, a novel skeletal muscle-specific member of the 10 calsarcin family, interacts with multiple Z-disc proteins. J Biol Chem 277, 13998-14004 11 (2002). 12 44. Faulkner, G. et al. ZASP: a new Z-band alternatively spliced PDZ-motif protein. J Cell 13 Biol 146, 465-475 (1999). 14 45. Arber, S. et al. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural 15 organization, dilated cardiomyopathy, and heart failure. Cell 88, 393-403 (1997). 16 46. Chowrashi, P., Mittal, B., Sanger, J.M. & Sanger, J.W. Amorphin is phosphorylase; 17 phosphorylase is an alpha-actinin-binding protein. Cell Motil Cytoskeleton 53, 125-135 18 (2002). 19 47. Rakus, D., Mamczur, P., Gizak, A., Dus, D. & Dzugaj, A. Colocalization of muscle 20 FBPase and muscle aldolase on both sides of the Z-line. Biochem Biophys Res Commun 21 311, 294-299 (2003). 22 48. Branon, T.C. et al. Efficient proximity labeling in living cells and organisms with 23 TurboID. Nat Biotechnol 36, 880-887 (2018). 24 49. Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in 25 organizing cellular architecture and function. Cell 131, 174-187 (2007). 26 50. Martin, K.C. & Ephrussi, A. mRNA localization: gene expression in the spatial 27 dimension. Cell 136, 719-730 (2009). 28 51. Boncompagni, S. et al. Mitochondria are linked to calcium stores in striated muscle by 29 developmentally regulated tethering structures. Mol Biol Cell 20, 1058-1067 (2009). 30 52. Ferreira, R. et al. Subsarcolemmal and intermyofibrillar mitochondria proteome 31 differences disclose functional specializations in skeletal muscle. Proteomics 10, 3142- 32 3154 (2010). 33 53. Regue, L. et al. IMP2 Increases Mouse Skeletal Muscle Mass and Voluntary Activity by 34 Enhancing Autocrine Insulin-Like Growth Factor 2 Production and Optimizing Muscle 35 Metabolism. Mol Cell Biol 39 (2019). 36 54. Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells 37 by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat Protoc 8, 38 162-175 (2013). 39 55. Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 40 253-268 (2015). 41 56. Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem cells. 42 Nature 504, 282-286 (2013). 43 57. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple 44 and accurate approach to expression proteomics. Mol Cell Proteomics 1, 376-386 (2002).

33 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license.

1 58. McAlister, G.C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed 2 detection of differential expression across cancer cell line proteomes. Anal Chem 86, 3 7150-7158 (2014). 4 59. Kutner, R.H., Zhang, X.Y. & Reiser, J. Production, concentration and titration of 5 pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4, 495-505 (2009). 6 60. Boudou, T. et al. A microfabricated platform to measure and manipulate the mechanics 7 of engineered cardiac microtissues. Tissue Eng Part A 18, 910-919 (2012). 8

34 Figure 1 Actinin-BirA* iPSC-CMs Focal adhesion a b c Actinin d complex Z-discs ⍺-Actinin 2 BirA* HA Control BirA* ECM 130 — Interacting protein Cytosol Actinin Z M +Biotin Proximal protein 100 — Distal protein 37 — GAPDH

Sarcomere Cortical actin ⍺-Actinin 2 BirA* HA ⍺ -Actinin 2 ~10 nm

e Input anti-HA IP f g Actinin h Actinin-BirA* enriched (n=285) Actinin Actinin Actinin-BirA* iPSCs Control BirA* Control−1 3 Control BirA*-HA Control BirA*-HA +Biotin +Biotin Cardiomyocyte differentiation 130 — HA Control−3 2 170 — 100 — Actinin Actinin-BirA* iPSC-CMs Control 130 — Control−2 1 25 — TCAP (Z) Biotin-free media 100 — ActininBirA3 3 BirA*

Myomesin (M) +50 µM Biotin pulse (24 hours) 70 — - 170 — ActininBirA2 2 (Intensity+1) 2 Log 55 — Protein extraction 40 — ActininBirA1 1 Actinin 35 — Streptavidin-IP 25 — Min Log2(Intensity) Max Quantitative Proteomics (TMT) 15 — Streptavidin-HRP

i Sarcomere j Focal Adhesion k Cell Junction l Metabolism m Mitochondrial membrane NEB CNN2 CTNNA1 PFKP MYPN FN1 ENAH ACTA2 SND1 HK1 SLC25A4 TCAP ACTN3 VCL CTNNB1 CTNNA3 VDAC2 PFKM PYGB DMD SPTAN1 SUCLG1 TNNI1 SORBS1 PGD LDB3 MLLT4 TLN1 TJP2 PKP2 ATP5A1 ACTN2 TPM1 VIM PGM1 SLC25A6 ZYX PALLD PYGM MYOZ2 MYL3 LMOD1 TJP1 TUFM CRK UGP2 SLC25A3 ACTN2 CALD1 MYH6 LDHB TTN PXN ACTN4 CSRP3 MYL4 TPM1 ACTN1 MYH7 ACTN3 MYH9 Actinin-BirA*-3 Actinin-BirA*-2 Control-3 Control-1 Control-2 SMYD1 MYBPC3 CRKL GAB1 Actinin-BirA*-1

MYH10 UNC45B n RNA Binding mRNA metabolic process Translation

NACA NACA RPS11 RPL14 RPL14 SMG7 CCT8 RPS11 PCBP1 PCBP2 EEF1A2 RPL35A SMG7 RPL35A RPL23 RPS27A CCT8 NOP56 SERBP1 TUFM RPS27A DDX46 HNRNPK RPS3 XRN1 EIF4G1 EIF5 RPS14 HBS1L CCT5 RPS20 SERBP1 HNRNPA2B1 HBS1L RPS20 RTN4 RPS14 RPS3 DHX9 EIF5 CCT5 XRN1 EEF2 CSDE1 EIF4G1 RUVBL1 ABCF1 EEF2 EEF1A2 RPL23 FTSJ3 RUVBL1 FTSJ3 NOP56 TUFM Figure 2

a Z-bodies cTnT-KO Actinin-BirA* iPSC-CMs

Actinin Streptavidin Merge

b Sarcomere assembly enriched (n=80) c Control−1 2 1.5 CSRP3 Control−3 1

Control−2 Control 3 1.0 MYH7 Control − 2 KO−2 2 Control − 1 KO−1 1 KO 0.5 cTnT

Control − 3 BirA* cTnT − KO Actinin − BirA* − 2 KO- −3 3 cTnT-KO cTnT − WT Actinin − BirA* − 3 WT3 3 cTnT − WT Actinin − BirA* − 1 WT2 0.0 Actinin 2 cTnT − WT Actinin − BirA* − 2 WT MYH10 Fold enrichment over cTnT − KO Actinin − BirA* − 1 WT1cTnT TLN2 cTnT − KO Actinin − BirA* − 3 1 -0.5 0 5 10 15 0 100 200 300 Max Min Log2(Intensity) Rank

d Glycolysis Sarcomere PFKM FLNA PGAM1 LDHB FLNC Translation PKM FILIP1 MYOZ2 RPL23 ZYX SMYD1 RPL35A CTTNBP2NL SVIL CSRP3 RPS14 NEXN SYNPO TTN RPS11 CTTNBP2 MYPN EEF1A2 ACTN4 LDB3 CCT5 CTTN CTNNA1 MYL3 MYBPC3 RPS20 HSPB1 PDLIM1 TPM1 TUBB6 MYH7 HSP90AA1 HSPA8 CLTC MYH6 TNNI1 TUBB4B SORBS1 HSPA1A MLLT4 UGP2 HSPA5 DDX46 ACTA2 HSP90B1 TUBB Unfolded PFN1 PGD SLC25A6 protein response (UPR) VDAC2 IDH2 NNT SLC25A3 Mitochondrial NAD/NADH binding membrane 10 Figure 3 5 TNNT2 Genotype RIP-Seq Overview a 0 b ControlInput RIP c endoplasmic reticulum Actinin-BirA* iPSC-CMs ActininPoly -BirA* RIP catalytic complex GO Gene Ratio GO Gene Ratio 0.30 Actinin-BirA*-depleted Actinin-BirA*-enriched −5 chromatin envelope 0.30 Biotin-free media 20 transcripts (1113) transcripts (945) chromosome 0.25 40 mitochondrial envelope 0.25

PC2: 11% variance 0.20 +50 µM Biotin pulse (24 hours) cytoskeletal part 0.20 15 mitochondrial membrane part −10 10 15 0.15 0.15 −20 0 20 microtubule cytoskeleton mitochondrial part 0.10 TNNT2 Genotype 12

Native RIP – cell lysis FDR PC1: 85% variance 0 nuclear body mitochondrial protein complex Actinin−BirAHA 10 9 −log (FDR) nuclear chromosome −log (FDR) 10 10 mitochondrion Streptavidin-IP Control -log 6 50 −10 nucleolus 60 3 organelle inner membrane 60 Actinin-BirA* proximity proteins nucleoplasm part 80 and bound transcripts 0 ribosomal subunit 70 PC2: 31% variance 100 −20 spliceosomal complex -6 -4 -2 0 2 4 6 80 ribosome RNA isolation −30−20−10 0 10 20 Log2FC over Control PC1: 40% variance

RNA-sequencing + analysis d e f g 3 1.0 1 Actinin NDUFA1 NDUFA1 TTN FC FC 2 0.5 FC 0 2 2 2 TTN 1 0.0 -1

0 -0.5 -2

-1 -1.0 -3 Average Gene Log Gene Average Average Gene Log Gene Average Average Gene Log Gene Average

-2 -1.5 -4 10 I II III IV V Glycolysis Thin Thick 5 ETC Complexes Sarcomere TNNT2 Genotype filaments 0 InputInput h p<0.01 i j PolysomePoly k TTN 60 2.0 −5 672 NDUFA1 10

50 NDUFA8 PC2: 11% variance p<0.05 GPX4 1.5 80S −10 273 5 40 −20 0 20 TNNT2 Genotype 1.0 PC1: 85% variance 833 30 0 Input Poly 20 40S ns Polysomes p<0.001 0.5 60S −5 10 UV Absorbance (A260) Enriched transcripts PC2: 11% variance % mRNA puncta per cell 0 0.0 −10 Actinin-BirA* RIP-Seq 15% Sucrose 50%

late endosome membrane 3.5 l ↓Actinin-BirA* depleted Polysome RNA-Seq mitochondrion 4.0 ↑Actinin-BirA* enriched (Log FC) ↑Active translation4.5 2 organellar ribosome ↑Active translation n=87 n=273 ribonucleoprotein complex GO Gene Ratio GO Gene Ratio −log (FDR) 2.0 catalytic complex 10 ribosome 0.3 envelope 2.5 0.20 ESCRT complex inner mito. membrane complex spliceosomal complex 3.00.2 0.15 late endosometroponin membrane complex 3.5 1.5 mitochondrial envelople 0.1 0.10 4.0 U2 type spliceosomalmitochondrion complex mitochondrial membrane part 4.5 organellar ribosome mitochondrial part −log (FDR) 1.0 −log (FDR) ribonucleoproteincatalytic complexcomplex 10 10 GO Gene Ratio mitochondrial protein complex 2.5 25 ESCRT ribosomecomplex 0.3 mitochondrion 3.0 30

late endosomespliceosomal membrane complex 3.50.2 0.5 organelle inner membrane 35 troponinmitochondrion complex 4.0 respirasome 40 0.1 -3 -2 -1 0 1 2 3 4.5 45 U2 type spliceosomalorganellar ribosome complex respiratory chain complex Actinin-BirA* RIP-Seq (Log2FC) 50 ribonucleoprotein complex GO Gene Ratio

ribosome 0.3

spliceosomal complex 0.2 troponin complex 0.1 U2 type spliceosomal complex Figure 4 a Input Streptavidin-IP b Input anti-HA IP Input Streptavidin-IP cTnT cTnT cTnT cTnT Actinin Actinin cTnT cTnT cTnT cTnT Control KO WT Control KO WT Control BirA*-HA Control BirA*-HA Control KO WT Control KO WT 70 — IGF2BP2 70 — IGF2BP2 70 — IGF2BP2 37 — 37 — PCBP1 PCBP1 37 — PCBP1 c d KH Actinin e Actinin 37 — Actinin IGF2BP2 PCBP2 37 — PCBP2domains37 — domains PCBP2 “Bait” ? domains ? “Prey” “Bait” “Prey” 55 — SERBP1 55 — SERBP1 55 — SERBP1 1-160 161-600 1-275 276-750 751-895 IGF2BP2 Actinin RRM KH Actin Binding (AB) Rod Calmodulin-like (CaM) 8 5 IGF2BP2 **** **** **** 4 **** 6 3 4 2 Merge 2 1

0 0 Relative M2H Luciferase Signal Relative M2H Luciferase Signal

Actinin Negative IGF2BP2 Negative AB domain KH domains Rod domainCaM domain RRM domains + Actinin + IGF2BP2 KH domains

10 µm Figure 5

shScrambleshIGF2BP2 a shScramble shIGF2BP2 b Actinin-BirA* proximity c Global transcript levels 70 — IGF2BP2 transcript levels shScramble shScramble 37 — GAPDH shScramble shIGF2BP2 shScramble shIGF2BP2 shIGF2BP2 shIGF2BP2 1.2 1.2 1.2 1.2 1.2 1.0 1.0 1.0 0.8 0.8 0.8 GAPDH 0.8 0.8* GAPDH GAPDH 0.6 0.6 * GAPDH * 0.6 * 0.4 ** 0.4 0.4 0.4 0.4 0.2 0.2 0.2 Relative to Relative to Relative to GAPDH Relative to 0.0 0.0 Relative to 0.0 0.0 0.0 shScramble shIGF2BP2 NDUFA1 NDUFA8 COX6B1 NDUFA11 NDUFA1 NDUFA8 NDUFA11 COX6B1 shScrambleshIGF2BP2 NDUFA1 NDUFA8 NDUFA11 NDUFA1 NDUFA8 NDUFA11 shScramble shScramble d shIGF2BP2 e f shIGF2BP2 1.2 1.2 1.0 shScramble shScramble1.2shIGF2BP2 1.0 shIGF2BP2 V 1.0 0.8 50 — 1.0 III 0.8 * * 0.8 37 — 0.8 ** 0.6 GAPDH II 0.6 0.6 ** 0.8 0.4 25 — IV 0.4 0.4 0.6 0.4 I 0.2 0.2 0.2

Relative to GAPDH 0.4 70 — Relative MitoTracker Relative to IGF2BP2 0.0

0.0 0.0 OCR (pmol/min/ng protein) 0.0 0.2 0 20 40 60 80 37 — GAPDH 0.0 Minutes shScramble OCR (pmol/min/ng protein) 0 20 40 60 80 NDUFA1 NDUFA8 Complex I NDUFA11shIGF2BP2Complex II Complex III Complex IV Complex V shScrambleshIGF2BP2 Minutes ETC Components

* g shScramble h iPSC-CMs i shIGF2BP2 2.0 1.2 1.2 1.0 + shScramble or 1.5 0.8 * * shIGF2BP2 0.8 ** Max. twitch 1.0 GAPDH 0.6 force 0.4 + 0.5 0.4 Relative OCR Fibroblasts ECM slurry 0.2 Relative Twitch Force (pmol/min/ng protein) Relative to 0.0 0.0 0.0

NDUFA1 NDUFA8 NDUFA11 shScramble shScrambleshIGF2BP2 Max respirationATP production Spare capacity Basal respiration shIGF2BP2 Non-mitochondrial

Sarcomere j shScramble k shIGF2BP2 1.2 ATP shScramble1.2shIGF2BP2 1.0 220 — β-MHC ⍺-actinin 2 0.8 OXPHOS RBP Nucleus 25 — 0.8 TnI GAPDH 0.6 0.4 70 — 0.4 IGF2BP2 0.2 Relative to GAPDH 37 — Relative to GAPDH ETC transcripts 0.0 0.0 Sarcomere/ TnI β -MHC chromatin transcripts UPR NDUFA1 NDUFA8 NDUFA11

Translation Extended data 1 a b c 0 µM 0.0016 0.008 0.04 0.2 1.0 5.0 50.0 Donor STOP Vector BirA* HA EF1 HygroR 170 — 800 bp 800 bp 3 ns 130 — gRNA+Cas9 100 — ACTN2 ACTN2 70 — Locus Exon 20 Exon 21 3’ UTR 2 55 — Streptavidin-HRP STOP 40 — 35 — Target ACTN2 STOP Exon 20 Exon 21 BirA* HA EF1 HygroR 25 — Integration 1 15 — Expressed ⍺-Actinin 2 BirA* HA Protein

Relative Twitch Force 0 37 — GAPDH

Control

Actinin-BirA*

d MAP4 NEBL XIRP1 TLN2 PKP2 MYPN SORBS2 PDLIM1 LPP MAGED2 NEB MYOZ2 CNN3 LDB3 CTNNA3 CAMK2D CKM TTN TNNI1 DLG1 FLNC MAP1B PALLD ACTN2 SND1 CSRP3 MYBPC3 MYL4 RDX TANC1 UTRN MYL3 CALD1 ATP2A2 TCAP OBSCN TJP1 ACTN1 DMD MYH7 NEXN PPP1R9B VCL MYH6 ATP1A1 DST SMYD1 FHL2 PPP1CA MACF1 CTNNA1 ACTN3 TJP2 CRKL MYH10 MAPRE2 EZR TLN1 LMOD1 SMTN SVIL TPM1 SYNRG PXN CRYAB FN1 INPPL1 ACTN4 PPP1R12A ARFGAP2 YWHAQ ENAH FLNA SLC8A1 BAG3 SYNPO2 TUBB6 SORBS1 MYH9 MLLT4 VIM MKL2 CTTNBP2NL TAGLN CLTC PICALM COPB1 UNC45B ARF3 FILIP1 TACC2 CTNNB1 ANK2 FERMT2 ACTA2 CSNK1A1 ZYX SH3D19 CTTNBP2 ITSN1 PFN1 NES LMO7 SYNPO CKAP5 CD2AP CTTN DPYSL4 CLINT1 GAB1 DYNC1H1 PRKAR2A CORO1C AAK1 NACA UBR4 ANK3 HSPA5 TUBA1B CRK SPTAN1 NUP214 RAN CCT6A DVL3 CCT8 CORO1B TUBB SPTBN1 EPS15L1 CFL1 DPYSL3 HSPB1 TUBB4B CNN2 DDX46 MRE11A TCP1 HSP90AB1 CAPZB RPL23 RUVBL1 RCSD1 ANXA6 DBN1 RTN4 HNRNPA2B1 HSPA8 UBA1 ANXA2 HSP90AA1 CCT5 HSP90B1 PGM1 DHX9 STAT3 TTC28 HSPA1A CSDE1 RPS3 PYGM PCBP2 FBXO30 PFKM RPS20 RPS27A RPL14 AHNAK ABCF1 PYGB USP15 DDX3X HNRNPK RPS11 RPL35A IDH1 PCBP1 NOP56 UGP2 LDHB MYO18A EEF1A2 SERBP1 EIF4ENIF1 EIF4G1 EIF5 EEF2 PGD RPS14 PKM HK1 PFKP LARP1 TUFM ATP5A1 HBS1L TNKS1BP1 NNT SUCLG1 IDH2 TNRC6B SMG7 EIF4G3 VCPIP1 SLC25A6

EIF4G2 FTSJ3 PRMT1 FASN XRN1 PGAM1

UBAP2L SLC25A4 SUCLA2 GCN1L1 SLC25A3 VDAC2 Extended data 2

a b Control Actinin-BirA* c d +Biotin +Biotin 2.0 200 cTnT-KO cTnT-WT cTnT cTnT cTnT cTnT cTnT ** KO WT KO WT KO WT 1.5 150 37 — —cTnTanti-cTnT 100 — —Actininanti-Actinin170 — 1.0 100 130 — 37 — GAPDH— anti-GAPDH 100 — 0.5 50

70 — OCR (pmol/min)

55 — Relative Mitotracker 40 — 0.0 0 35 — KO WT 0 20 40 60 80 25 — cTnT Minutes 15 — Streptavidin-HRP e cTnT-KO 4 cTnT-WT *** *** 3 *** ***

2 ****

1

Relative OCR (pmol/min) 0

Max RespirationATP Production Spare Capacity Basal RespirationNon-Mitochondrial 0.5 1 1.5 Extended data 3 1 2 1 2 1 2 1.5 1 0.5 0 − − − 2 1 0 − − 2 1 0 − − 2 1 0 − − 1 2

a Complex I 2 II 1 0 III − − IV V COX1 COX2 COX3 COX4I1 COX5A COX5B COX6A2 COX6B1 COX6C COX7A1 COX7A2 COX7A2L COX7B COX7C COX8A NDUFA4 ATP5A1 ATP5B ATP5C1 ATP5D ATP5E ATP5F1 ATP5G1 ATP5G2 ATP5G3 ATP5H ATP5I ATP5J ATP5L ATP5O ATP6 SDHA SDHB SDHC SDHD CYB UQCR10 UQCRB UQCRC1 UQCRC2 UQCRFS1 UQCRH UQCRQ NDUFA1 NDUFA2 NDUFA8 NDUFA10 NDUFA11 NDUFAB1 NDUFB1 NDUFB2 NDUFB3 NDUFB4 NDUFB5 NDUFB6 NDUFB7 NDUFB8 NDUFB9 NDUFC1 NDUFC2 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFS8 NDUFV1 NDUFV3 4 Actinin−BirA*Actinin RIP−−BirA*4 RIP−Actinin4 −BirA* RIP−4 Actinin−BirA* RIP−4 ActininLog2(TPM)−BirA* RIP−4

BirA* Gene Z-score - 3 Actinin−BirA*Actinin RIP−−BirA*3 RIP−Actinin3 −BirA* RIP−3 Actinin−BirA* RIP−3 NDUFA1Actinin2 −BirA* RIP−3 NDUFA2 2 Actinin−BirA*Actinin RIP−−BirA*2 RIP−Actinin2 −BirA* RIP−2 Actinin−BirA* RIP−2 NDUFA3Actinin−BirA* RIP−2 NDUFA5 NDUFA6 1 Actinin 1 Actinin−BirA*Actinin RIP−−BirA*1 RIP−Actinin1 −BirA* RIP−1 Actinin−BirA* RIP−1 NDUFA7Actinin−BirA* RIP−1 NDUFA8 4 Control RIPControl−4 RIP−4 Control RIP −4 Control RIP−4 NDUFA9Control0 RIP−4 NDUFA10 3 Control RIP−3 NDUFA11 Control RIP−3 Control RIP −3 Control RIP−3 NDUFAB1Control RIP−3 NDUFB1 −1 2 Control RIPControl−2 RIP−2 Control RIP−2 NDUFB2 Control Control RIP −2 Control RIP−2 NDUFB3 1 Control RIPControl−1 RIP−1 NDUFB4 Control RIP −1 Control RIP−1 NDUFB5Control−2 RIP−1 NDUFB6 1 2 NDUFB7

2 1 0 − − NDUFB8 NDUFB9 NDUFC1 NDUFC2 b Glycolysis NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFS7 NDUFS8 NDUFV1 NDUFV3 Control RIP Control RIP Control RIP Control RIP Actinin Actinin Actinin Actinin AAAS ALDOA ARNT DHTKD1 ECD ENO1 ENO2 ENO3 GALK1 GAPDH GPI HDAC4 HIF1A HK1 HK2 INSR JMJD8 LDHA NCOR1 NUP107 NUP133 NUP153 NUP155 NUP160 NUP188 NUP205 NUP210 NUP214 NUP35 NUP37 NUP43 NUP50 NUP85 NUP88 NUP93 NUP98 OGDH OGDHL OGT PFKFB2 PFKFB3 PFKFB4 PFKL PFKM PFKP PGAM1 PGK1 PGM1 PKM PPARA PRKAA1 PRKAG1 PRKAG2 RAE1 RANBP2 SEC13 STAT3 TPI1 TPR ZBTB20 − − − −

4 BirA* RIP BirA* RIP BirA* RIP BirA* RIP ActininLog2(TPM)−BirA* RIP−4 BirA* − − − − - 3 ActininGene1 Z2 -score3 4 −BirA* RIP−3

NDUFA1 2 − − − − 2 NDUFA2Actinin−1 BirA*2 3 4 RIP−2 NDUFA3

Actinin 1 NDUFA5 NDUFA6Actinin1 −BirA* RIP−1 NDUFA7 4 NDUFA8Control RIP−4 NDUFA9 0 3 NDUFA10 NDUFA11Control RIP−3 NDUFAB1 2 NDUFB1 −1 Control NDUFB2Control RIP−2 NDUFB3 1 NDUFB4 NDUFB5Control−2 RIP−1 NDUFB6 NDUFB7 NDUFB8 NDUFB9

1 NDUFC1 NDUFC2 1 0 − 1 NDUFS1 c 1 0 − d NDUFS2 NDUFS3 Thin filament Thick filament Control Elution NDUFS4 Enriched Actinin-BirA* Elution Control ElutionNDUFS5 Enriched Actinin-BirA*NDUFS6 Elution Depleted Actinin-BirA* Elution NDUFS7 NDUFS8 Depleted Actinin-BirA*NDUFV1 Elution NDUFV3 4 **** 4 Control RIP Control RIP Control RIP Control RIP Actinin Actinin Actinin Actinin **** 3 3 **** **** − − − − **** BirA* RIP BirA* RIP BirA* RIP BirA* RIP TNNC1 TNNI1 TNNI3 TNNT1 TNNT2 TPM1 TPM2 TPM3 MYBPC3 MYH6 MYH7 MYL2 MYL3 MYL4 MYOM2 OBSCN TTN ** 2 ** ** **

Log2(TPM) 2 − − − −

GAPDH ** *** 1 2 3 4 4 Actinin−BirA* RIP−4 Gene Z-score 1 ** 1

Actinin−BirA* RIP−4 − − − − 1 2 3 4 BirA*

- 0 3 Actinin−BirA* RIP−3 TNNC1Actinin−BirA* RIP0 −3 FC TPM

TNNC2 2 -1 2 Actinin−BirA* RIP−2 TNNI1Actinin−BirA* RIP-1 −2 1 ** -2 ****

TNNI2 Log **** Actinin 1 -2 Actinin−BirA* RIP−1 TNNI3Actinin−BirA* RIP−1 **** -3 FC relative to

TNNT1 2 4 Control RIP−4 TNNT2Control0 RIP−4 -3 -4 TNNT3 3 -4 Log Control RIP−3 TPM1Control RIP−3 -5 TPM2 −1 2 TPM3 Control Control RIP−2 Control RIP−2 H19 MYBPC3 TTN H19 TTN 1 GPX4 TPM1 GPX4 TPM1 Control RIP−1 MYH6Control RIP−1 RPS19 RPS19 NDUFA1NDUFA8 NDUFA1NDUFA8 MYH7 NDUFA11 HIST1H1E NDUFA11 MYL2 HIST1H1E MYL3 RIP-Seq qPCR MYL4 MYOM1 MYOM2 OBSCN TTN e Control RIP Control RIP Control RIP Control RIP Actinin fActinin Actinin Actinin g − − − − BirA* RIP BirA* RIP BirA* RIP BirA* RIP − − − −

Actinin 1 2 3 4 − − − −

NDUFA1 1 2 3 4 3.0 0.0 NDUFA8

FC) ↑Actinin-BirA* enriched ↓Actinin-BirA* depleted 2 FC) 2.5 NDUFB1 ↑Active translation 2 -0.5 ↑Active translation n=273 n=87 NDUFA1 2.0 NDUFAB1 -1.0 MYL4 TNNC1 1.5 NDUFB5 -1.5

NDUFS6 NDUFA11 TNNC2 NDUFA8

1.0 NDUFB9 -2.0 NDUFB6 Actinin-BirA* RIP-Seq (Log

NDUFS3 Actinin-BirA* RIP-Seq (Log -2.5 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 Polysome RNA-Seq (Log FC) Polysome RNA-Seq (Log2FC) 2 Extended data 4 Input anti-HA IP Actinin Actinin Control BirA*-HA Control BirA*-HA Input Input anti-HA IP a Streptavidin-IP b c 8 cTnT cTnT cTnT cTnT 70 — Actinin Actinin IGF2BP2 **** Actinin ? RBPs Control KO WT Control KO WT Control BirA*-HA Control BirA*-HA “Bait” “Prey” 6 37 — PCBP1 7037 — IGF2BP2PCBP1

37 — PCBP2 37 — PCBP2 4 37 — PCBP1 55 — SERBP1 55 — SERBP1 37 — PCBP2 2

55 — SERBP1 0 d e Relative M2H Luciferase Signal Actinin IGF2BP2 ? Negative PCBP1 PCBP2 Method “Bait” “Prey” IGF2BP2 SERBP1 RNA 8 + Actinin Binding TMT SP-IP HA-IP M2H **** Protein 6 IGF2BP2 + + + + 4 PCBP1 + + + - PCBP2 + + + - 2 SERBP1 + + - - 0 Relative M2H Luciferase Signal

ACTN1 ACTN2 ACTN3 Negative ACTN4 + IGF2BP2 Extended data 5 a Actinin-BirA* iPSC-CMs IgG IP 3 IGF2BP2 IP Biotin-free media * +50 µM Biotin pulse * * (24 hours) 2 * GAPDH

Cell lysis

1

IgG-IP IGF2BP2-IP Normalized to

0 IGF2BP2 peptide pulldown

NDUFA1 NDUFA8 NDUFA11 COX6B1 Streptavidin IP

RNA isolation + quantitative PCR

b Total cells Live cells Single cells Analysis (78.0%) (93.9%) (87.6%) 100K 150K 100K

Single Cells 87.6 80K 80K 60

100K 60K 60K Total Cells 78.0 40 Count FSC-A SSC-A Live Cells 40K 93.9 40K 50K Hoechst Blue-H 20

20K 20K

0 0 0 0 3 3 4 5 0 50K 100K 150K 3 3 4 5 0 50K 100K 150K 200K 250K -10 0 10 10 10 -10 0 10 10 10

SSC-A Alexa Fluor 647-A Hoechst Blue-A GFP-A :: Mitotracker

Specimen_001_IMP2shRNA1_001.fcs Specimen_001_IMP2shRNA1_001.fcs Specimen_001_IMP2shRNA1_001.fcs Specimen_001_IMP2shRNA1_001.fcs Ungated Total Cells Live Cells Single Cells 5000 3898 3662 3208