Hnrnpa2b1 in Normal Skeletal Muscle Formation and Regeneration

hnRNPA2B1 in Normal Skeletal Muscle Formation and Regeneration

Oscar Whitney

Committee Members Bradley Olwin, Ph.D., Department of Molecular, Cellular, and Developmental Biology Jennifer Martin, Ph.D., Department of Molecular, Cellular, and Developmental Biology Nicole Stob, Ph.D., Department of Integrative Physiology

Undergraduate Thesis Defense on April 4th, 2019

Research Advisor: Bradley Olwin, Ph.D. Day-to-day Mentor: Thomas Vogler, Ph.D.

University of Colorado Boulder Department of Molecular, Cellular, and Developmental Biology

Table of Contents

I. Abstract

II. Introduction

III. Results

IV. Discussion

V. Materials and Methods

VI. References

I. Abstract

Skeletal muscle is essential for our everyday mobility and has the unique ability to regenerate after injury through its resident muscle stem cells (MuSCs) (Frontera, Ochala 2015,

Almeida et al., 2016). When the muscle is injured, its MuSCs transition to a myoblast cell fate, proliferate and fuse to each other and into injured muscle to create and repair skeletal muscle

(Wang, Rudnicki, 2012). MuSCs express many new genes required for muscle formation that are challenging to transcribe, splice and translate into protein (Bland et al., 2010). We do not yet fully understand how nascent skeletal muscle regulates new mRNAs during muscle formation.

However, we do know that regenerating and forming skeletal muscle expresses many RNA- binding proteins (RNABPs), which may regulate mRNA during myogenesis (Apponi et al.,

2011, Keene et al. 2007). I sought to identify RNABPs that regulate mRNA and elucidate their function in skeletal muscle formation. I identified that the expression of the RNABP hnRNPA2B1 is up-regulated in the nuclei of MuSCs in a mouse model of skeletal muscle regeneration. Although hnRNPA2B1 is not necessary for skeletal muscle myoblast proliferation, it supports skeletal muscle differentiation and formation. Subsequently, I found that hnRNPA2B1 binds splicing machinery mRNA which then splice myogenic transcripts. These data indicate that hnRNPA2B1 regulates mRNA splicing to support skeletal muscle differentiation and formation. Further, these findings point towards RNABPs as regulators of mRNA stability and splicing during skeletal muscle differentiation.

II. Introduction

Skeletal muscle is essential for our everyday life, as it allows us to perform tasks

as simple as getting out of bed. If we injure our skeletal muscle, it can regenerate itself using

skeletal muscle stem cells (MuSCs) (Frontera, Ochala 2015, Almeida et al., 2016). MuSCs reside

close to the muscle fiber and are quiescent in uninjured muscle (Wang, Rudnicki, 2012).

Quiescent MuSCs express the transcription factor Pax7 and do not proliferate (Wang, Rudnicki,

2012). Quiescent MuSCs activate in response to skeletal muscle injury and become myoblasts,

which proliferate to expand the pool of MuSCs (Wang, Rudnicki, 2012).

Figure 1. Skeletal muscle stem cells (MuSCs) from quiescence to muscle formation

Schematic describes MuSC differentiation from quiescence to self-directed fusion or muscle fiber directed fusion. Quiescent MuSCs are activated, become myoblasts and turn on the transcription factor MyoD. Myoblasts proliferate and express Pax7 and MyoD. Myoblasts differentiate, turn off Pax7 and express MyoD and Myogenin. Differentiated myoblasts fuse to each other or to injured skeletal muscle fibers to create or repair muscle. (Wang, Rudnicki, 2012, Frontera, Ochala 2015, Almeida et al., 2016)

Myoblasts express the transcription factors MyoD and Myogenic factor 5 (Wang,

Rudnicki, 2012). These myoblasts next differentiate into myocytes by expressing the skeletal muscle differentiation inducing transcription factor Myogenin and downregulating the MuSC transcription factor Pax7 (Wang, Rudnicki, 2012). Differentiated myocytes fuse into injured muscle fibers or to each other to repair or create new skeletal muscle fibers (Wang, Rudnicki,

2012) (Fig 1.).

New muscle fibers are large, multi-nucleated syncytia that contain a contractile apparatus

(Frontera, Ochala 2015, Almeida et al., 2016). Differentiating myoblasts must transcribe, regulate, splice and translate the mRNA needed to differentiate into myocytes and then build a muscle fiber’s contractile apparatus. We do not yet understand how myoblasts process the mRNA of myogenic transcription factors, like that of Myogenin, or the mRNA of large contractile unit proteins, like that of Titin. We speculate that RNA-binding proteins (RNABPs) splice, stabilize and transport these mRNAs in newly forming muscle fibers. For example, the

RNABP TDP-43 interacts with Titin mRNA, an essential component of the muscle contractile unit, to presumably protect and transport it (Vogler et al., 2018). In binding Titin mRNA, TDP-

43 re-localizes to the cytoplasm of skeletal muscle to transport Titin to its site of translation

(Vogler et al., 2018). We do not know whether other RNABPs undergo a similar cytoplasmic relocation to transport or regulate mRNA during muscle formation.

RNABPs present in MuSCs and myoblasts regulate mRNA splicing, which may facilitate the alternative splicing shift differentiating muscle undergoes (Bland, 2010). One of these

RNABPs, hnRNPA2B1, is expressed in many tissues, including skeletal muscle (ProteomicsDB,

2019). Mutated hnRNPA2B1 is found in toxic cytoplasmic protein aggregates in the skeletal muscle of patients with multisystem proteinopathy (MSP) (Kim et al., 2013). In MSP, mutations to hnRNPA2B1’s C-terminal low-complexity domain increase its aggregation potential, leading to cytoplasmic aggregates that contain hnRNPA2B1 (Kim et al., 2013). hnRNPA2B1 aggregates are also found in patients with amyotrophic lateral sclerosis (ALS) (Martinez et al., 2016), limb- girdle muscular dystrophy (LGMD) (Bengoechea et al., 2014) and oculopharyngeal muscular dystrophy (OPMD) (Fan and Rouleau, 2003). Cytoplasmic aggregates of hnRNPA2B1 are thought to be a key driver of disease-associated neuromuscular degeneration (Kim et al., 2013).

As aggregated hnRNPA2B1 is present in neuronal disease, most work on hnRNPA2B1 has focused on its neuronal function. In neurons, hnRNPA2B1 modulates alternative splicing and binds mRNA via its UAGG RNA-binding motif to splice, polyadenylate, transport and export it.

Further, hnRNPA2B1 processes and packages micro-RNAs into exosomes in immune cells (Fig

2.) (Glisovic et al., 2008; Keene, 2007; Villaroya-Beltri et al., 2013; Martinez et al., 2016;

Nguyen et al., 2018).

Although aggregates of mutant hnRNPA2B1 are present in neurons and muscle cells affected by a diverse range of disease, the role of hnRNPA2B1 in healthy tissue remains poorly understood. Characterization of hnRNPA2B1 in neurons led to the hypothesis that mutations in the hnRNPA2B1 low-complexity domain caused cytosolic mislocalization and aggregation of hnRNPA2B1.

Fig 2. Known hnRNPA2B1 functions in neurons and immune cells Schematic of previously discovered hnRNPA2B1 functions. hnRNPA2B1 binds to RNA to regulate its splicing, polyadenylation and transport in neurons. hnRNPA2B1 is also processes & sorts micro-RNA into exosomes in immune cells. (Glisovic et al., 2008; Keene, 2007; Villaroya-Beltri et al., 2013; Martinez et al., 2016; Nguyen et al., 2018)

Cells are affected by these mutations in two ways: (1) hnRNPA2B1 aggregation impairs

the normal nuclear function of hnRNPA2B1 to interact with RNA and (2) Low-complexity

domain mutations increase the aggregation of hnRNPA2B1, resulting in the formation of

cytotoxic amyloid-like aggregates (Kim et al., 2013; Martinez et al., 2016). These studies have broadened our understanding of the RNAs bound to hnRNPA2B1 in neurons and the role of hnRNPA2B1 in disease. However, the functional roles of hnRNPA2B1 and a detailed description of its RNA-binding targets in skeletal muscle are unknown.

A paralog of hnRNPA2B1, hnRNPA1, is an essential RNABP that splices RNA and is also found in pathological aggregates located in diseased muscle tissue (Liu et al., 2016).

Knockout of hnRNPA1 is embryonically lethal to developing mouse pups due to severe cardiac muscle defects. In heterozygous hnRNPA1 knockout mice, hnRNPA2B1 does not compensate for the loss of hnRNPA1, suggesting that these two proteins may have distinct functions in skeletal muscle (Liu et al., 2017). Understanding the role of hnRNPA2B1 in skeletal muscle will allow us to understand how RNABPs regulate muscle differentiation and how RNABP-related diseases affect muscle differentiation and regeneration. Therefore, I explored the basic function of hnRNPA2B1 in wild type skeletal muscle.

To determine the role of hnRNPA2B1 in skeletal muscle, I analyzed the expression and protein levels of hnRNPA2B1 within MuSCs and skeletal muscle fibers in muscle sections from wild-type mice. Subsequently, I knocked out hnRNPA2B1 from C2C12 myoblasts to determine effects caused by hnRNPA2B1 loss. I chose the C2C12 murine myoblast model of skeletal muscle, an immortalized MuSC line, to model MuSC behavior in-vitro. These C2C12 myoblasts proliferate in serum-rich culture and express the transcription factors MyoD, Myf5 and Pax7

(Burattini, 2004). Subsequently, C2C12 myoblasts can imitate muscle formation as they can be driven to differentiate and fuse to each other to create skeletal muscle fiber precursors

(myotubes) (Burattini, 2004).

I found that hnRNPA2B1 protein is prevalent and mRNA expression is up-regulated in the nuclei of MuSCs and skeletal muscle fibers in regenerating skeletal muscle. Subsequently, I found that a loss of hnRNPA2B1 in C2C12 myoblasts impairs their differentiation into skeletal muscle but does not impair their proliferation. I then assessed the RNA partners of hnRNPA2B1 in proliferating and differentiated myotubes by performing an enhanced UV crosslinking and immunoprecipitation assay and RNA-sequencing (Van Nostrand et al., 2016). I found that hnRNPA2B1 binds to a subset of mRNA that regulate splicing. Thus, hnRNPA2B1 regulates splicing gene mRNA and a loss of hnRNPA2B1-mediated splicing regulation may be the cause for the observed differentiation defect. Impairment of differentiation caused by hnRNPA2B1 loss-of-function may cause part of the muscle degeneration seen in diseases where hnRNPA2B1 is mutated or aggregated.

III. Results

The cytoplasmic localization and aggregation of hnRNPA2B1, similar to that of related

RNABPs such as TDP-43, is a pathological hallmark of several degenerative skeletal muscle

diseases (Bengoechea et al., 2015; Fan and Rouleau, 2003; Kim et al., 2013). Although TDP-43

is found in cytoplasmic aggregates in diseased muscle, recent data demonstrate that TDP-43

cytoplasmic localization is critical for normal muscle formation (Vogler et al., 2018). Thus,

hnRNPA2B1 re-localization to the cytosol may also be a part of normal muscle regeneration. To

test this, I examined the subcellular distribution of hnRNPA2B1 in uninjured tibialis anterior

muscle and chemically injured tibialis anterior muscle after five, ten and thirty days of

regeneration (Caldwell et al., 1990) (Fig. 3.1.).

Figure 3.1. Tibialis Anterior injury and regeneration timeline. Tibialis anterior (TA) muscles were isolated from 3-6-month old male and female mice (C57Bl/6 strain) following BaCl2 induced injury (Caldwell et al., 1990). TA were collected 5, 10 and 30 days after injury.

In uninjured tibialis anterior muscle, hnRNPA2B1 localizes to the nuclei of all skeletal

muscle cells that express hnRNPA2B1, which is similar to the localization of the related RNABP

TDP-43 in uninjured muscle (Figure 3.1a). hnRNPA2B1 remains localized to the nucleus five

days after injury, and unlike TDP-43, does not appear in the cytoplasm (Figure 3.2b, c).

a. Laminin, DAPI Laminin, Pax7 Laminin, A2B1 Merged

Uninjured

5 DPI 5

10 DPI 10

30 DPI 30

Figure 3.2. Localization of hnRNPA2B1 during skeletal muscle regeneration. (a-d) Immunoreactivity for hnRNPA2B1 during skeletal muscle regeneration in vivo. (a) hnRNPA2B1 localization in the myonucleus (arrow) and MuSC (arrowhead) in uninjured muscle. (b) hnRNPA2B1 localization in the myonucleus (arrow) and MuSC (arrowhead) in 5 days post injury (DPI) muscle. (c) hnRNPA2B1 localization in the myonucleus (arrow) and MuSC (arrowhead) in 10 DPI muscle. (d) hnRNPA2B1 localization in the myonucleus (arrow) and MuSC (arrowhead) in 30 DPI muscle. All images were scaled above secondary antibody generated background. Nuclei counterstained with DAPI. Scale bars are 25 µm.

Although hnRNPA2B1 localization does not change during regeneration, the majority of

centrally located nuclei express hnRNPA2B1. The presence of hnRNPA2B1 protein in MuSC

nuclei and in the central nuclei of muscle fibers during muscle regeneration suggests that it may

be important for the formation of new muscle.

Single-cell sequencing of the tibialis anterior MuSC transcriptome after 4 and 7 days of

regeneration reveals that hnRNPA2B1 transcript expression increases 26-fold in Pax7-expressing

MuSCs (Fig 3.3.) (Data generated by Bradley Pawlikowski). These data further support that

hnRNPA2B1 is upregulated in MuSCs to support skeletal muscle regeneration.

Figure 3.3. hnRNPA2B1 transcript expression in Pax7+ MuSCs during skeletal muscle regeneration. (a) hnRNPA2B1 transcript fold-change in skeletal muscle Pax7+ MuSCs in uninjured muscle and after 4 and 7 days post-injury (DPI). Transcript levels obtained via single-cell RNA sequencing of all Tibialis anterior tissue cells in uninjured muscle and muscle after 4 and 7 DPI. Fold-change calculated via CellRanger, normalized to unique molecular identifiers added to sequencing as barcode tags.

Given that hnRNPA2B1 transcript expression increases during skeletal muscle regeneration, I suspected that hnRNPA2B1 regulates RNA during muscle formation. However, it is unknown which RNAs hnRNPA2B1 binds to during skeletal muscle formation. Thus, we characterized the set of hnRNPA2B1 bound RNAs in myoblasts and multinucleated myotubes using enhanced CLIP (eCLIP) with the help of my collaborator Eric Nguyen (Figure 4.1a) (Van

Nostrand et al., 2016). hnRNPA2B1 eCLIP peaks highly correlated between biological replicates and identified known hnRNPA2B1 mRNA targets, including hnRNPA2B1’s own 3’ UTR

(Figure 4.1b-d). Similar to hnRNPA2B1 binding sites in neurons (Martinez et al., 2016), hnRNPA2B1 peaks were enriched in the 3’ UTR of RNAs in both myoblasts and myotubes

(Figure 4.1e). Figure 4. on next page.

a. b.

c. d.

e. Myoblasts Myotubes

Figure 4.1. Enhanced CLIP reveals hnRNPA2B1 interacts primarily with the 3’ UTR of RNA in skeletal muscle. (a) Schematic for hnRNPA2B1 enhanced CLIP in during skeletal muscle differentiation. (b) Myoblast and myotube eCLIP are highly correlated for significant peaks (defined as significantly enriched over size -8 matched input, p<10 . (c) and (d) Representative gene tracks in both myoblasts and myotubes for hnRNPA2B1 reveals hnRNPA2B1 interacts with its own RNA transcript through binding to its own 3’UTR. (e) Location of peaks in hnRNPA2B1 RNA targets reveals a strong bias towards 3’UTR interactions

hnRNPA2B1 and TDP-43 are similar RNA-binding proteins as they both harbor low- complexity domains, RNA-binding motifs and are both associated with skeletal muscle diseases

(Vogler et al., 2018; Glisovic et al., 2008; Kim et al., 2013). However, hnRNPA2B1 localizes to the nucleus of regenerating skeletal muscle (Fig. 3.2.), whereas TDP-43 re-localizes to the cytoplasm of regenerating skeletal muscle (Vogler et al., 2018). This localization difference led me to ask whether hnRNPA2B1 and TDP-43 bind to similar or distinct RNA transcripts during muscle formation. While hnRNPA2B1 and TDP-43 share RNA targets, the majority of transcripts bound are unique to each of these two RNABPs, suggesting that hnRNPA2B1 regulates a distinctly different class of RNAs than TDP-43 (Figure 4.2 a, b). hnRNPA2B1 interacts with RNAs involved in alternative splicing and splice site selection, whereas TDP-43 interacts with transcripts that encode contractile apparatus proteins (Vogler et al., 2018) (Table

1). hnRNPA2B1’s nuclear localization and upregulation along with its splicing mRNA interactions suggest that it regulates splicing in the nucleus of myogenic cells to support muscle formation.

Figure 4.2. Enhanced CLIP reveals hnRNPA2B1and TDP-43 mostly interact with distinct RNAs in muscle. (a) Overlap of RNA targets in myoblasts between TDP-43 and hnRNPA2B1. (b) Overlap of RNA targets in myotubes between TDP-43 and hnRNPA2B1.

Table 1. GO term analysis of RNAs that interact with hnRNPA2B1 in myotubes Analysis of RNA transcripts identified by eCLIP on hnRNPA2B1 from n = 2 biological replicates.

To examine the effects of hnRNPA2B1 loss during skeletal muscle formation, I created an hnRNPA2B1 knockout (KO) C2C12 myoblast cell line using CRISPR-Cas9 (aided by Joshua

Wheeler). I transfected three C2C12 myoblasts clones with a Cas9 gene, one of two distinct hnRNPA2B1-guide RNA and a Puromycin resistance gene construct via liposomal cell delivery.

I transfected another three C2C12 myoblast clones with a Cas9 gene, scrambled-sequence guide

RNA to control for off-target Cas9 cuts and a Puromycin resistance gene (Figure 5.a).

hnRNPA2B1 sgA2B1 #1 hnRNPA2B1 sgA2B1 #2 sgScrambled CRISPR-Cas9 CRISPR-Cas9 CRISPR-Cas9 Transfection Transfection Transfection

Seven days of puromycin treatment to select for stably transfected, construct containing myoblasts

b. sgRNA Scrambled A2B1 #1 A2B1 #1 A2B1 #2

hnRNPA2B1 rabbit pAB

hnRNPA2B1 mouse mAB

GAPDH

c. hnRNPA2B1 mouse monoclonal AB d. hnRNPA2B1 rabbit polyclonal AB

A2B1 DAPI, A2B1 A2B1 DAPI, A2B1

A2B1 WT A2B1

A2B1 KO A2B1 A2B1 KO A2B1

Figure 5. Knockout of hnRNPA2B1 in C2C12 myoblasts. (a) Schematic for hnRNPA2B1 knockout in C2C12 myoblasts using CRISPR-Cas9. Two distinct guides targeting the hnRNPA2B1 locus were used. A scrambled guide RNA was used as a control. (b) Western blot analysis of hnRNPA2B1 protein in hnRNPA2B1 specific guide treated myoblasts (KO) and hnRNPA2B1 control guide treated myoblasts (WT). Two separate antibodies were used to probe for hnRNPA2B1: a rabbit polyclocal antibody (pAB) and a mouse monoclonal antibody (mAB). GAPDH served as a loading control. c. hnRNPA2B1 immunoreactivity using the mouse monoclonal antibody in KO and control cells. d. hnRNPA2B1 immunoreactivity using the rabbit polyclocal antibody in KO and control cells Nuclei counterstained with DAPI. Scale bars are 20 µm. 17

C2C12 myoblasts stably integrated the hnRNPA2B1 knockout guide RNA and scrambled guide RNA, as assessed by Puromycin selection. hnRNPA2B1 knockout efficiency was variable between hnRNPA2B1 sgRNAs; however, hnRNPA2B1 sgRNA #1 effectively reduced hnRNPA2B1 protein levels to nearly undetectable levels as compared to cells transfected with a scrambled small guide RNA (sg Scrambled) (Figure 5.b). I isolated three separate clones from cells transfected with hnRNPA2B1 sgRNA #1. Next, I assessed these clones’ hnRNPA2B1 protein levels via immunoreactivity using a monoclonal and a polyclonal antibody against hnRNPA2B1. Both antibodies clearly showed reduced protein levels by Western blot, meaning that the hnRNPA2B1 sgRNA #1 successfully knocked out hnRNPA2B1 (Figure 5.b). Only the monoclonal hnRNPA2B1 antibody detected a knockout of hnRNPA2B1 protein by immunoreactivity in the hnRNPA2B1 C2C12 clones (Figure 5.c). The polyclonal hnRNPA2B1 antibody recognized a nuclear antigen regardless of hnRNPA2B1 knockout (Figure 5.d). Thus, the specificity of the polyclonal antibody for hnRNPA2B1 protein is low.

As hnRNPA2B1 knockdown decreases cancer cell proliferation (Barceló et al., 2014;

Golan-Gerstl et al., 2011), I sought to determine whether hnRNPA2B1 similarly reduced proliferation in hnRNPA2B1 knockout myoblasts. I treated hnRNPA2B1 sgRNA myoblasts

(hnRNPA2B1 KO) and Scrambled sgRNA myoblasts (hnRNPA2B1 WT) with 5-ethynyl-2’- deoxyuridine (EdU) to mark DNA-replicating (proliferating) cells (Figure 6.a, b). Surprisingly, hnRNPA2B1 loss had no effect on myoblast proliferation (Figure 6.c). Thus, hnRNPA2B1 is not necessary for C2C12 myoblast proliferation

DAPI, EdU A2B1 Merged

A2B1 WTA2B1

A2B1 KO

p=0.14

Figure 6. Knockout of hnRNPA2B1 in C2C12 myoblasts does not affect proliferation. (a) Experimental outline of 5’-ethynyl-2’-deoxyuridine (EdU) proliferation assay. Myoblasts were treated with 10mM EdU for 3 hours, fixed and immunoreacted for hnRNPA2B1, EdU and DAPI. (b) EdU Representative images of hnRNPA2B1 knockout (KO) and control (WT) myoblasts treated with EdU for 3 hours. (c) Quantification of C2C12 myoblasts in (a) reveals non-significant difference (as defined by p>0.05) between hnRNPA2B1 KO and scrambled control myoblasts. Scale bars are 50 µm.

As hnRNPA2B1 loss does not affect myoblasts proliferation, I assessed whether a related

RNABP is compensating for hnRNPA2B1 loss. I thus assessed whether the structurally related

RNABP hnRNPA1 compensates for hnRNPA2B1 loss, via immunoreactivity to hnRNPA1. I

observed that the intensity of hnRNPA1 immunoreactivity did not differ between hnRNPA2B1

control and knockout cells (Figure 7.a) leading to the preliminary conclusion that hnRNPA1

expression is not upregulated to compensate for hnRNPA2B1.

a. DAPI hnRNPA1 Merge

A2B1 WTA2B1

A2B1KO

Figure 7. Knockout of hnRNPA2B1 is not compensated for by upregulation of hnRNPA1. (a) Representative images of hnRNPA2B1 knockout and control cells after 24 hours of differentiation. Cells chemo- reacted against DAPI (row 1), immunoreacted against hnRNPA1 (row 2) and DAPI and hnRNPA1 reactivity merged (row 3). hnRNPA1 immunoreactivity is comparable between hnRNPA2B1 WT and KO cells, implying no gross change of hnRNPA1 due to hnRNPA2B1 loss.

Given that hnRNPA2B1 expression is upregulated 26-fold in MuSC after 4 days of skeletal muscle regeneration, I presumed that hnRNPA2B1 plays a role in skeletal muscle differentiation and regeneration. Therefore, I examined how hnRNPA2B1 loss affects myoblast differentiation. hnRNPA2B1 knockout myoblasts that had differentiated for 48 hours failed to make large multinucleated myotubes (Figure 8.1a) and instead formed smaller myotubes containing fewer nuclei than control myotubes (Figure 8.1b, c). hnRNPA2B1 knockout myoblasts were delayed in expressing the protein myosin heavy chain (MyHC), which is required for myoblasts to form mature muscle, and the myogenic transcription factor Myogenin

(MyoG), which is required for myoblasts to differentiate into myocytes. These findings imply that myoblasts’ loss of hnRNPA2B1 induces a defect in differentiation rather than fusion, as the myoblasts can fuse, but form smaller myotubes (Figure 8.1d, e). Therefore, hnRNPA2B1 loss impairs myoblast differentiation and skeletal muscle formation.

As assessed via preliminary Western blot data, hnRNPA2B1 knockout myoblasts contain less MyHC protein after 3 days of differentiation as compared to hnRNPA2B1 control myoblasts

(Figure 8.2a). hnRNPA2B1 knockout myoblasts appear to contain slightly lower levels of

Myogenin protein than hnRNPA2B1 control myoblasts after 3 days of differentiation, implying that hnRNPA2B1 loss does not ablate Myogenin protein expression (Figure 8.2b). Further, hnRNPA2B1 knockout myoblasts appear to contain more Myogenin protein than hnRNPA2B1 control myoblasts after 4 days of differentiation, implying that Myogenin protein levels attain normal levels a day later in hnRNPA2B1 knockout myoblasts (Figure 8.2c).

a. Day 2 Myotubes: MyHC, DAPI

Knockout Control

b. c.

* * *

d. e.

* * *

Figure 8.1. Knockout of hnRNPA2B1 inhibits myogenic differentiation. (a) Representative images of hnRNPA2B1 knockout and control cells after 48 hours of differentiation. Myotubes stained with myosin heavy chain (MyHC) and nuclei counterstained with DAPI. (b) 72-hour time- course shows a delay in the fusion index in hnRNPA2B1 KO cells. (c) 72-hour time-course shows a delay in the number of nuclei in myosin heavy chain (MHC)-expressing cells in hnRNPA2B1 KO cells (d) 72- hour time-course shows a delay in the number of cells22 expressing MyHC in hnRNPA2B1 KO cells. (e) 72- hour time-course shows a delay in the number of cells expressing myogenin (MyoG) in hnRNPA2B1 KO cells. Controls are sgScrambled C2C12 cells.

a. MyHC, Day 3 Myotubes

A2B1 KO A2B1 KO A2B1 KO sgRNA WT Clone 1 WT Clone 2 WT Clone 4 Clone1 Clone 2 Clone 4

myHC f59 antibody

GAPDH control

b. MyoG, Day 3 Myotubes

A2B1 KO A2B1 KO A2B1 KO sgRNA WT Clone 1 WT Clone 2 WT Clone 4 Clone1 Clone 2 Clone 4

MyoG Rab antibody

Tubulin control

c. MyoG, Day 4 Myotubes A2B1 KO A2B1 KO A2B1 KO WT Clone 1 WT Clone 2 WT Clone 4 sgRNA Clone1 Clone 2 Clone 4

MyoG F5D antibody

GAPDH control

Figure 8.2. Knockout of hnRNPA2B1 inhibits myogenic differentiation. (a) Western blot analysis of myosin heavy chain expression of hnRNPA2B1 WT and KO clones after 72 hours of differentiation. Second row of blots represents GAPDH loading control (b) Western blot analysis of Myogenin expression of hnRNPA2B1 WT and KO clones after 72 hours of differentiation. Second row of blots represents Tubulin loading control (c) Western blot analysis of Myogenin expression of hnRNPA2B1 WT and KO clones after 96 hours of differentiation. Second row of blots represents GAPDH loading control.

In summary, hnRNPA2B1 is up-regulated in the nuclei of MuSCs in regenerating muscle

(Fig 3.2, 3.3) and binds splicing genes’ mRNA (Fig 4.1, 4.2) during skeletal muscle regeneration and formation. hnRNPA2B1 loss impairs myoblasts’ differentiation (Fig 8.1) but does not affect their proliferation (Fig 6.). Therefore, hnRNPA2B1 appears to mediate skeletal muscle differentiation and formation by regulating splicing genes that direct alternative splicing in the nucleus.

IV. Discussion

I. The role of hnRNPA2B1 in regulating skeletal muscle differentiation

Here, I show that hnRNPA2B1 is up-regulated in MuSCs and present in the nuclei of

MuSCs and skeletal muscle during normal skeletal muscle regeneration. During myogenesis, hnRNPA2B1 binds primarily to the 3′ UTR of a distinct class of RNAs that regulate splicing in skeletal muscle. hnRNPA2B1 knockout impairs skeletal muscle differentiation and formation but does not alter myoblast proliferation. These data demonstrate a link between hnRNPA2B1 splicing mRNA binding function and skeletal muscle differentiation. Further investigation is required to understand how hnRNPA2B1 loss affects its bound mRNA partners to impair differentiation. Loss of hnRNPA2B1 reducing the expression or translation of key splicing regulators during muscle differentiation would explain why hnRNPA2B1 knockout impairs muscle differentiation and formation.

Given that hnRNPA2B1 loss impairs but does not completely block differentiation, it is plausible that RNAs normally bound by hnRNPA2B1 act combinatorically to drive skeletal muscle differentiation. Absence of hnRNPA2B1 could cause subtle loss-of-function in these

splicing mRNA, which would prevent them from splicing myogenic mRNA necessary for skeletal muscle differentiation (Bland et al., 2010). Our eCLIP experiment shows that hnRNPA2B1 interacts with the MEF2a gene transcript in myotubes, which is a transcription factor that is alternatively spliced during muscle differentiation and drives the expression of myogenic transcripts necessary for muscle differentiation (Nakka et al., 2018). Thus, loss of hnRNPA2B1 may affect MEF2a transcript splicing and MEF2a protein presence. hnRNPA2B1 may also be supporting differentiation by regulating the transcripts of DDX5 and DDX17

(transcripts bound by hnRNPA2B1 in our eCLIP data), which act as a splicing-enhancers during terminal differentiation of skeletal muscle (Dardenne et al., 2014). DDX5 and DDX17 specifically regulate MyoD, a gene that maintains myoblast cell fate (Tapscott et al., 1988).

Continuing experimentation to explore and validate how hnRNPA2B1 regulates RNA targets of splicing will bring about a deeper understanding of the mechanism by which hnRNPA2B1 supports muscle formation.

Given that hnRNPA2B1 knockout cells experience a differentiation defect but express comparable levels of the myogenic regulator MyoG to control myoblasts, it is possible that hnRNPA2B1 loss affects very early regulators of differentiation. Investigating the expression of early myogenic factors in hnRNPA2B1 knockout cells may provide an insight into the mechanistic basis by which hnRNPA2B1 regulates myoblast differentiation.

II. Divergent roles of the related RNABPs hnRNPA2B1 and TDP-43 in muscle

hnRNPA2B1 and TDP-43 are related RNA-binding proteins that share similar RNA- binding and low-complexity domain motifs, regulate RNA splicing and both aggregate in diseased tissues (Bengoechea et al., 2015; Kim et al., 2013; Kuo et al., 2009; Martinez et al.,

2016; Neumann et al., 2006; Wu et al., 2018). However, these two RNA-binding proteins appear to have distinct roles in skeletal muscle formation (Vogler et al., 2018). hnRNPA2B1 binds transcripts involved in splicing, whereas TDP-43 interacts with large transcripts that encode contractile apparatus proteins (Vogler et al., 2018). TDP-43 re-localizes to the cytoplasm during muscle regeneration and forms complexes with large RNAs, whereas hnRNPA2B1 remains in the nucleus during muscle regeneration and is not associated with granule aggregates (Vogler et al., 2018). The knockout of hnRNPA2B1 has no effect on the proliferation of myoblasts, whereas the knockout of TDP-43 causes cell death of myoblasts (Vogler et al., 2018). Another notable difference between hnRNPA2B1 and TDP-43 are their distinct RNA-binding motifs (UAGG and

UG-rich, respectively) (Martinez et. al, 2016, Vogler et. al, 2018.). This may be why hnRNPA2B1 and TDP-43 bind different sets of RNA, which may in turn mediate hnRNPA2B1 and TDP-43’s distinct functions. To understand RNABP mediated mRNA regulation in forming skeletal muscle we need to understand how these two seemingly similar RNA-binding proteins perform such different functions.

III. The role of hnRNPA2B1 function in disease

Our experiments on hnRNPA2B1 underscore several broader principles relating to involvement of RNABPs in skeletal muscle formation, maintenance and disease. First, cytosolic localization and aggregation of RNABPs is believed to be a key driver of disease in several degenerative diseases of the central nervous system and skeletal muscle (Bengoechea et al.,

2015; Kim et al., 2013; Neumann et al., 2006; Weihl et al., 2008). Cytoplasmic RNABP localization is considered pathogenic, but recent TDP-43 experiments suggest that the redistribution of RNABPs in disease is likely a consequence of a heightened requirement for

normal cellular repair (Glisovic et al., Vogler et al., 2018). As such, we suggest that additional

RNABPs, which have been classically described as mislocalized, might in fact be reflecting a normal cellular response upregulated in disease by the increased need for regeneration and repair. However, not all RNABPs re-localize to the cytosol of regenerating skeletal muscle.

During skeletal muscle injury, hnRNPA2B1 remains in the nucleus and interacts with the transcripts of nuclear proteins. Thus, the localization and function of other RNA-binding proteins that localize to the cytoplasm of tissues in degenerative disease (e.g. TIA1, Matrin-3, hnRNPA1, hnRNPDL) warrant case-by-case examinations during skeletal muscle regeneration.

Since hnRNPA2B1 remains localized to the nucleus in healthy skeletal muscle, hnRNPA2B1’s cytoplasmic mislocalization in disease likely alters its normal functions in skeletal muscle (Kim et al., 2013). Therefore, cytoplasmic localization of hnRNPA2B1 in disease could impair myogenic mRNA splicing in the nucleus, thus disrupting skeletal muscle differentiation. Over time, this could have degenerative effects on skeletal muscle.

Understanding the impact of hnRNPA2B1 cytoplasmic mis-localization on affected skeletal muscle’s differentiation potential and RNA splicing ability will elucidate the muscle- degenerative effects of aggregative diseases. Together with protein aggregates’ inherent cytotoxicity, additional RNABP loss-of-function may explain why cytoplasmic RNABP aggregates cause skeletal muscle degeneration.

V. Methods

Mice

Mice were bred and housed according to National Institutes of Health (NIH) guidelines for the ethical treatment of animals in a pathogen-free facility at the University of Colorado at

Boulder. The University of Colorado Institutional Animal Care and Use Committee (IACUC) approved all animal protocols and procedures. Wild-type mice were C57Bl/6 (Jackson Labs,

ME, USA). Tibialis anterior (TA) muscles were isolated from 3-6-month-old male and female wild-type mice. Control mice were age and sex matched from the mice and crosses described above.

Mouse Injuries

Mice at 3-6 months old were anesthetized with isofluorane, the left TA muscle was injected with 50μL of 1.2% BaCl2, and then the injured and contralateral TA muscles were harvested at the indicated time points.

Immunofluorescence staining of tissue sections

TA muscles were dissected, fixed on ice for 2hrs with 4% paraformaldehyde, and then transferred to PBS with 30% sucrose at 4°C overnight. Muscle was mounted in O.C.T. (Tissue-

Tek®) and cryo-sectioning was performed on a Leica cryostat to generate 10μm thick sections.

Tissues and sections were stored at -80°C until staining. Tissue sections were post-fixed in 4% paraformaldehyde for 10 minutes at room temperature (RT) and washed three times for 5 min in

PBS. Immunostaining with anti-Pax7, anti-Laminin and anti-hnRNPA2B1 antibodies required

heat-induced epitope retrieval where post-fixed slides were placed in citrate buffer, pH 6.0, and subjected to 6 min of high pressure-cooking in a Cuisinart model CPC-600 pressure cooker

(Tockman et al., 1998). For immunostaining, tissue sections were permeabilized with 0.25%

Triton-X100 (Sigma) in PBS containing 2% bovine serum albumin (Sigma) for 60 min at RT.

Incubation with primary antibody occurred at 4°C overnight followed by incubation with secondary antibody at room temperature (RT) for 1hr. Primary antibodies included mouse anti-

Pax7 (Developmental Studies Hybridoma Bank, University of Iowa, USA) at 1:750, rabbit anti- laminin (Sigma-Aldrich) at 1:200, rabbit anti- hnRNPA2B1 (Abcam) at 1:200 and mouse anti- hnRNPA2B1 (Abcam) at 1:200. Alexa secondary antibodies (Molecular Probes) were used at a

1:1000 dilution. Sections were incubated with 1 μg/mL DAPI for 10 min at room temperature then mounted in Mowiol supplemented with DABCO (Sigma-Aldrich) or ProLong Gold

(Thermo) as an anti-fade agent.

Microscopy and image analyses

Images were captured on a Nikon inverted spinning disk confocal microscope. Objectives used on the Nikon were: 10x/o.45NA Plan Apo, 20x/0.75NA Plan Apo and 40x/0.95 Plan Apo.

Confocal stacks were projected as maximum intensity images for each channel and merged into a single image. Brightness and contrast were adjusted for the entire image as necessary. Images were processed using Fiji ImageJ.

hnRNPA2B1 eCLIP-seq

C2C12 myoblasts were seeded at 6 x 106 cells per 15 cm plate, grown 24hrs at 37C, 5%

CO2 and either harvested (undifferentiated myoblasts) or differentiated in differentiation media

for 2 days. hnRNPA2B1 enhanced CLIP (eCLIP) was performed according to established protocols (Van Nostrand et al., 2016). In brief, hnRNPA2B1-RNA interactions were stabilized with UV crosslinking (254 nm, 150mJ/cm2). Cell pellets were collected and snap frozen in liquid

N2. Cells were thawed, lysed in eCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1%

NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1x protease inhibitor) and sonicated

(Bioruptor). Lysate was RNAse I (Ambion, 1:25) treated to fragment RNA. Protein-RNA complexes were immunoprecipitated using indicated antibody. One size-matched input

(SMInput) library was generated per biological replicate using an identical procedure without immunoprecipitation. Stringent washes were performed as described, RNA was dephosphorylated (FastAP, Fermentas), T4 PNK (NEB), and a 3’ end RNA adaptor was ligated with T4 RNA ligase (NEB). Protein-RNA complexes were resolved on an SDS-PAGE gel, transferred to nitrocellulose membranes, and RNA was extracted from membrane. After RNA precipitation, RNA was reverse transcribed using SuperScript IV (Thermo Fisher Scientific), free primer was removed, and a 3’ DNA adapter was ligated onto cDNA products with T4 RNA ligase (NEB). Libraries were PCR amplified and dual-indexed (Illumina TruSeq HT). Pair-end sequencing was performed on Illumina NextSeq sequencer.

Cell culture.

C2C12 cells: Immortalized murine myoblasts (American Type Culture Collection) were maintained on uncoated standard tissue culture plastic or gelatin-coated coverslips at 37°C with

5% CO2 in DMEM with 20% fetal bovine serum and 1% penicillin/streptavidin. To promote myoblast fusion and differentiation when the C2C12 cells reached confluence, they were

switched to 5% horse serum, 1% penicillin/streptavidin and 1% Insulin-Transferrin-Selenium in

DMEM.

hnRNPA2B1 CRISPR-Cas9 knockout and EdU incorporation

CRISPR-Cas9 knockout was performed in C2C12 myoblasts. Single guide RNA

(sgRNA) against hnRNPA2B1 were designed using (crispr.mit.edu) and cloned into pSpCas9(BB)-2A-Puro (PX459). C2C12 myoblasts were transfected with JetPrime using standard protocols. Myoblasts were selected with puromyocin (1 g/mL) for one week. C2C12 myoblasts were incubated with 10µM 5-ethynyl-2'-deoxyuridine (EdU – Life Technologies) for two hours. Cells were washed, fixed and stained. For analysis that included EdU detection, EdU staining was completed prior to antibody staining using the Click-iT EdU Alexa fluor 488 detection kit (Molecular Probes) following manufacturer protocols.

Biochemical characterization of hnRNPA2B1 during myogenesis

In brief, C2C12 myoblasts and myotubes were lysed with RIPA buffer (50 mM Tris pH

7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl). Protein concentrations were determined using BCA assay (Thermo Scientific) according to standard procedures. Lysates were centrifuged at 18,000 x g for 20 minutes at 4C. Western blotting was performed following resolution of protein lysates on SDS-PAGE.

Immunofluorescence Staining of Cultured Cells

C2C12 cells were washed with PBS in a laminar flow hood and fixed with 4%

Paraformaldehyde for 10 min at room temperature in a chemical hood. Immunostaining for

hnRNPA2B1 requires heat-induced epitope retrieval where post-fixed cells were placed in citrate buffer, pH 6.0, and subjected to 6 min of steaming. Cells were then permeabilized with 0.25%

Triton-X100 in PBS containing 2% bovine serum albumin (Sigma) for 1 hour at RT. Incubation with primary antibody occurred at 4°C overnight followed by incubation with secondary antibody at room temperature for 1hr. Primary antibodies included mouse anti-hnRNPA2B1

(Abcam) at 1:200, rabbit anti-hnRNPA2B1 (Abcam) at 1:200, mouse anti-MyoG

(Developmental Studies Hybridoma Bank, University of Iowa, USA) at 1:270, hnRNPA1

(NovusBio) at 1:200, and a mouse anti-MHC (MF-20, Developmental Studies Hybridoma Bank,

University of Iowa, USA) at 1:1. Alexa secondary antibodies (Molecular Probes) were used at a

1:1000 dilution. All antibodies were diluted in with 0.125% Triton-X100 in PBS containing 2% bovine serum albumin. Cells were incubated with 1 μg/mL DAPI for 10 min at room temperature then mounted in Mowiol supplemented with DABCO (Sigma-Aldrich) as an anti-fade agent.

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