Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes

Abstract

RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3΄UTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.

The hnRNP RALY regulates transcription and cell proliferation by modulating the expression of specific factors including the proliferation marker E2F1

“…Interestingly, E2F1 and E2F2 mRNAs were identified previously as specific targets of RALY in the RIP-seq experiment performed in MCF7 cells, whereas all the proliferation repressors of the E2F family were not detected as RALY RNA interactors (Fig. 3B) (32).…”

“…To have a more comprehensive understanding of the general function of RALY on its target mRNAs, we intersected the list of differentially expressed genes detected in HeLa cells with the list of RALY targets identified previously by RIP-seq in MCF7 cells (32). We found that 193 up-regulated genes and 359 downregulated genes were present in both lists (Fig.…”

“…We recently identified RALY RNA targets by performing RIPseq analysis in MCF7 cells and studied the gene expression profile of RALY-silenced MCF7 cells. The two analyses identified 2929 RNAs bound by RALY and 217 differently expressed genes consequent to its down-regulation (32). Crossing these results, we selected 23 genes that are differently expressed after RALY silencing and for which the transcript is bound by RALY.…”

“…Among these targets, we focused on ANXA1 and H1FX, which were found, respectively, to be up-and down-regulated both at the RNA and the protein level upon RALY silencing. We also characterized the RNA-binding property of RALY by showing that it binds poly-U stretches, preferentially positioned in the 3Ј-UTR of protein-coding transcripts (32). Through proteomic analysis, RALY was found to interact with the C-terminal domain of RNAPII as well as with proteins involved in RNA metabolism and translational control, including polysomes (31)(32)(33).…”

“…We recently observed that the silencing of RALY in MCF7 cells caused the down-regulation of different genes related to cell cycle progression and that RALY-down-regulated cells showed a reduction in cell growth rate compared with control cells (32). Furthermore, transcripts coding for cell cyclerelated proteins were enriched among the RNA bound by RALY (32). To characterize the role of RALY in cell proliferation, we studied the gene expression profile of HeLa cells after RALY down-regulation.…”