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Changes in Mrna Abundance Drive Shuttling of RNA Binding Proteins, Linking 2 Cytoplasmic RNA Degradation to Transcription 3 Sarah Gilbertson1, Joel D

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Changes in Mrna Abundance Drive Shuttling of RNA Binding Proteins, Linking 2 Cytoplasmic RNA Degradation to Transcription 3 Sarah Gilbertson1, Joel D bioRxiv preprint doi: https://doi.org/10.1101/295972; this version posted September 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 under aCC-BY-ND 4.0 International license. 1 Changes in mRNA abundance drive shuttling of RNA binding proteins, linking 2 cytoplasmic RNA degradation to transcription 3 Sarah Gilbertson1, Joel D. Federspiel2, Ella Hartenian1, Ileana M. Cristea2, and Britt 4 Glaunsinger1,3,4* 5 1Department of Molecular and Cell Biology, University of California, Berkeley CA 94720-3370, 6 2Department of Molecular Biology, Princeton University, Princeton, NJ 08544, 3Department of 7 Plant & Microbial Biology, University of California, Berkeley CA 94720-3370, 4Howard 8 Hughes Medical Institute. 9 *Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/295972; this version posted September 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 under aCC-BY-ND 4.0 International license. 10 Abstract 11 Alterations in global mRNA decay broadly impact multiple stages of gene expression, although 12 signals that connect these processes are incompletely defined. Here, we used tandem mass tag 13 labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as 14 frequently occurs during viral infection, results in subcellular redistribution of RNA binding 15 proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression 16 of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including 17 poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the 18 localization or abundance of numerous factors linked to mRNA turnover. Using these data, we 19 uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing 20 recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our 21 results show that changes in cytoplasmic mRNA decay can directly impact protein localization, 22 providing a mechanism to connect seemingly distal stages of gene expression. 23 Introduction 24 mRNA decay is a critical stage of gene expression that regulates the abundance and 25 lifespan of cellular mRNAs. Many viruses including alpha and gammaherpesviruses, influenza A 26 virus, and SARS coronavirus accelerate host mRNA degradation through the use of viral proteins 27 that trigger endonucleolytic cleavage of mRNAs in the cytoplasm. Each of these viral proteins 28 bypasses the rate-limiting deadenylation step of the basal decay pathway, resulting in cleaved 29 mRNAs that are rapidly degraded by the major cellular 5’-3’ exonuclease Xrn1 (Covarrubias et 30 al., 2011; Gaglia et al., 2012). This process, termed ‘host shutoff’, allows viruses to rapidly 31 restrict cellular gene expression in order to blunt immune responses and liberate resources for 32 viral replication (Abernathy and Glaunsinger, 2015; Burgess and Mohr, 2015; Gaglia, 2010; 2 bioRxiv preprint doi: https://doi.org/10.1101/295972; this version posted September 25, 2018. 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 under aCC-BY-ND 4.0 International license. 33 Rivas et al., 2016). Viral endonucleases have also served as tools for deciphering how cells sense 34 and respond to large changes in mRNA abundance. 35 While mRNA decay is often considered the terminal stage of gene expression, the rate of 36 mRNA decay has recently been shown to influence transcription by RNA polymerase II 37 (RNAPII) in both yeast and mammalian cells (Abernathy et al., 2015; Braun and Young, 2014; 38 Haimovich et al., 2013; Sun et al., 2013). In yeast, a buffering system exists in which Xrn1 plays 39 a major role in connecting mRNA synthesis and decay, presumably allowing cells to maintain an 40 appropriate overall mRNA abundance. Mammalian cells also have a mechanism to sense mRNA 41 levels, though the pathway appears to operate differently than in yeast. Here, accelerated 42 cytoplasmic mRNA degradation does not lead to a compensatory increase in mRNA synthesis, as 43 might be predicted by the homeostatic model, but instead decreases cellular RNAPII promoter 44 recruitment, thereby amplifying the restrictive gene expression environment (Abernathy et al., 45 2015). Significant transcriptional repression as measured by nascent mRNA production was 46 reported to occur at approximately 9% of host genes, although validation experiments suggested 47 this number is likely to be an underestimate (Abernathy et al., 2015). 48 The mRNA decay-transcription feedback pathway is activated in mammalian cells 49 infected with gammaherpesviruses like Kaposi’s sarcoma-associated herpesvirus (KSHV) and 50 murine gammaherpesvirus 68 (MHV68), as well as upon expression of virally encoded mRNA 51 endonucleases in uninfected cells. Herpesviral endonucleases, including the muSOX protein of 52 MHV68, cleave mRNA but do not impact the abundance of noncoding RNAs transcribed by 53 RNA polymerase I (RNAPI) or III (RNAPIII) (Covarrubias et al., 2011). Correspondingly, 54 muSOX-induced mRNA decay elicits a significant decrease in RNA polymerase II (RNAPII) 55 recruitment to cellular promoters (Abernathy et al., 2015). Notably, depletion of Xrn1 from 3 bioRxiv preprint doi: https://doi.org/10.1101/295972; this version posted September 25, 2018. 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 under aCC-BY-ND 4.0 International license. 56 muSOX-expressing cells prevents the ensuing RNAPII transcriptional repression. This suggests 57 that the initial mRNA cleavage and translational inactivation are insufficient to restrict RNAPII 58 recruitment, and that subsequent exonucleolytic degradation of the cleaved mRNA fragments is a 59 critical signaling step. 60 Little is currently known about this pathway linking cytoplasmic mRNA decay to 61 RNAPII activity in mammalian cells, including the nature of the signal that is transmitted 62 between the two cellular compartments. An attractive hypothesis is that one or more RNA 63 binding proteins (RBPs) differentially traffics between the cytoplasm and the nucleus when basal 64 rates of mRNA decay are perturbed, thereby conveying global mRNA abundance information. 65 Recent analyses indicate that mammalian cells contain hundreds of RBPs that bind 66 polyadenylated mature mRNAs, and proteins within this group have been shown to regulate all 67 stages of gene expression (Gerstberger et al., 2014; Mitchell and Parker, 2014; Müller-McNicoll 68 and Neugebauer, 2013; Singh et al., 2015). Furthermore, RBPs frequently display 69 nucleocytoplasmic shuttling behavior. 70 Here, we charted global alterations in protein localization that occur specifically in 71 response to increased or decreased Xrn1 activity. This revealed a set of mammalian RBPs that 72 preferentially move from the cytoplasm to the nucleus during accelerated mRNA decay, as well 73 as components of the 5’-3’ decay machinery and other RBPs whose subcellular distribution is 74 altered in cells lacking Xrn1. Poly(A) tail associated proteins are overrepresented among the 75 RBPs that accumulate in the nucleus under conditions of global mRNA decay, offering an 76 explanation for how RNAPII could be selectively sensitive to mRNA abundance. Indeed, we 77 uncovered a new role for cytoplasmic poly(A) binding protein (PABPC) in mediating mRNA 78 decay-driven repression of RNAPII promoter recruitment. Furthermore, we show that the 4 bioRxiv preprint doi: https://doi.org/10.1101/295972; this version posted September 25, 2018. 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 under aCC-BY-ND 4.0 International license. 79 recruitment of TATA binding protein (TBP) to promoters is also impaired in response to PABPC 80 nuclear translocation, indicating that cytoplasmic mRNA decay impacts early events in 81 preinitiation complex assembly. Our results reveal how mRNA levels exert significant influence 82 on RBP localization and suggest that select RBPs transmit mRNA abundance information from 83 the cytoplasm to the nucleus to broadly influence gene expression, particularly under conditions 84 of cellular stress. 5 bioRxiv preprint doi: https://doi.org/10.1101/295972; this version posted September 25, 2018. 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 under aCC-BY-ND 4.0 International license. 85 Results 86 RNA binding proteins translocate from the cytoplasm to the nucleus in cells undergoing 87 enhanced cytoplasmic mRNA decay 88 To chart mRNA decay-driven movement of proteins between the cytoplasm and the 89 nucleus, we used a quantitative liquid chromatography/tandem mass spectrometry (LC/MS-MS)- 90 based approach. Specifically, following subcellular fractionation, proteins from nuclear and 91
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