Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

Abstract

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.

RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters

“…Consistent with the above analyses, MHV68 infected cells contained less promoter proximal Pol II than uninfected cells, and this signal was partially recovered in the R443I infection ( Fig 1E). This is consistent with data from previous studies that found no defect in serine 2 phosphorylation of elongating Pol II and no consistent change to Pol II elongation during MHV68 infection [23,25]. Notably, the difference in regression line slopes between MHV68 and mock infected cells was more marked when comparing Pol II promoter occupancy, suggesting that the primary transcriptional defect is at the stages of recruitment and initiation.…”

“…We previously showed that accelerated mRNA decay by the coordinated activity of muSOX and Xrn1 causes RNA binding protein relocalization from the cytoplasm to the nucleus and that this relocalization plays a role in transcriptional repression in 293T cells [23]. In agreement with our previous findings [23], we observed the expected increase in nuclear levels of the RNA binding proteins PABPC1 and LARP4 in cells infected with WT MHV68 compared to mock or R443I infected cells (Fig 3B). We then multiplexed these TMT labeled fractions and subjected them to quantitative liquid chromatography/tandem mass spectrometry (LC/MS-MS) (Figs 3A and S2A, S1 Table).…”

“…We previously reported that Pol II occupancy at several individual mammalian promoters was significantly reduced during accelerated cytoplasmic mRNA decay [23,25]. To more comprehensively assess the extent of mRNA decay-induced transcriptional repression, we evaluated global Pol II occupancy by chromatin immunoprecipitation and deep sequencing (ChIP-seq) in mock versus MHV68 infected MC57G mouse fibroblasts at 24 hours post infection (hpi), which is near the end of the infectious cycle [26].…”

“…We previously showed that accelerated mRNA decay by the coordinated activity of muSOX and Xrn1 causes RNA binding protein relocalization from the cytoplasm to the nucleus and that this relocalization plays a role in transcriptional repression in 293T cells [23]. We previously showed that accelerated mRNA decay by the coordinated activity of muSOX and Xrn1 causes RNA binding protein relocalization from the cytoplasm to the nucleus and that this relocalization plays a role in transcriptional repression in 293T cells [23].…”

“…Infection with the gammaherpesviruses KSHV or MHV68 causes reduced Pol II occupancy at individually tested host promoters in a manner dependent on mRNA cleavage by the viral endonucleases SOX or muSOX [22,23]. However, studies with mammalian RNA-decay inducing viruses and viral nucleases have revealed that accelerated mRNA decay is not broadly counteracted by increased transcription-based repopulation, but instead leads to a more extensive shutdown of cellular gene expression.…”