RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific
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ABSTRACT:
PROVIDER: PRJNA889324 | ENA |
REPOSITORIES: ENA
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Project description:Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)—proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.
2023-03-31 | GSE215198 | GEO
Project description:Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)-proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.
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Project description:Transcription factors (TFs) orchestrate the gene expression programs that define each cell’s identity, and the canonical TF accomplishes this with two functional domains1–7. The DNA-binding domain interacts with specific genomic sequences, and the effector domain binds coactivators or co-repressors that contribute to transcriptional regulation. We report here that TFs also frequently contain an RNA-binding domain and that this also contributes to gene regulation. Nearly half of TFs in human cells show evidence of RNA-binding and their affinities for RNA are similar to those of well-studied RNA-binding proteins. The RNA-binding sites of TFs have sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat8,9. RNA-binding contributes to TF function by promoting the dynamic association of TFs with chromatin. We conclude that the canonical definition of transcription factors is incomplete, and that the ability of many TFs to bind DNA, RNA and protein is fundamental to gene regulation.
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Project description:The RNA interactomes of HeLa and HEK293 cells jointly comprise 1106 RNA-binding proteins (RBPs), with almost half of these lacking well-defined RNA-binding domains (RBDs), suggesting the existence of numerous unknown RNA-binding architectures. Here, we report on “RBDmap”, a new method built on interactome capture, to comprehensively identify the RBDs of native RBPs in HeLa cells. Making use of in vivo UV-crosslinking of RBPs to polyadenylated RNAs, capture on oligo(dT) magnetic beads, sequential proteolytic digestion and LC-MS/MS combined with a sophisticated scoring algorithm, RBDmap “re-discoveres” many known RNA-binding sites (e.g. RRM, KH) of numerous well characterized RBPs and suggests novel RBDs.
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Project description:In the current work, we present a comprehensive coverage of the RNA-binding protein (RBP) interactome of Drosophila at four levels of resolution. The highest level tackled in our study represents the RNA-protein complexes, which we address by combining formaldehyde crosslinking of Drosophila S2 cells followed by polyA+ pulldown using oligo-dT beads. On the second level of resolution we identified direct RBPs in the cell using UV crosslinking. Given the paucity of information available on the RNA-binding regions in the Drosophila RBPs that we uncovered, we developed the CAPRI methodology to dissect RNA binding domains (RBDs) on a systematic level. The CAPRI technique provided the final two levels of resolution of our study. The protocol utilised FASP to enable isolation of peptides crosslinked to RNA and peptides adjacent to these crosslinked sites. The CAPRI pipeline mapped both the peptides in proximity to RNA and the precise amino acids contacting RNA. The RNA crosslinked peptide analysis was made by de novo peptide analysis software, PEAKS (Bioinformatics Solutions Inc). In a separate analysis, we also applied FA crosslinking to RBD capture and showed it to be a viable complementary method to the CAPRI workflow. We next applied CAPRI interactome capture to human cells in order to analyse the evolutionary conservation of newly identified RNA binding domains between the two species. These interactome capture techniques and the datasets acquired using them are discussed in detail in the following sections. In summary, we present a comprehensive resource for the study of RNA-protein interactions at a high throughput scale.
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