The kinetic landscape of an RNA binding protein in cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE150214 | GEO | 2020/12/16
REPOSITORIES: GEO
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Project description:RNAs are continuously associated with RNA-binding proteins (RBPs), and these interactions are necessary for many key cellular processes ranging from splicing to chromatin regulation. Although numerous approaches have been developed to map RNA-binding sites of individual RBPs, few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply this method to the HeLa transcriptome and compare RBP binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites. Protein interaction profile sequencing (PIP-seq) in HeLa cells. Two crosslinkers (formaldehyde and UV) with two RNases (dsRNase and ssRNase) each, as well as a no-crosslink sample. Performed with and without proteins. Three replicates for formaldehyde, two replicates for UV, single replicate for no crosslinker.
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Project description:RNAs are continuously associated with RNA-binding proteins (RBPs), and these interactions are necessary for many key cellular processes ranging from splicing to chromatin regulation. Although numerous approaches have been developed to map RNA-binding sites of individual RBPs, few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply this method to the HeLa transcriptome and compare RBP binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.
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Project description:Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cellspecific RBP, in miRNA biogenesis and cell proliferation. First, DAZL colocalized with miRNA let-7a in human PGCs and upregulated the levels of over a hundred mature miRNAs, including eight out of nine let-7 family, miR10, and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs upregulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline-tumor-cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing.
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2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress
Project description:Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions. Duplicate gPAR-CLIP libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. polyA RNAs were isolated for all conditions. Total RNA were isolated from log phase growth conditions. Sucrose gradient fractionation was performed: some RNAs were isolated from the "light" fraction (lighter than 40S ribosome) and some from the "heavy" fraction. gPAR-CLIP libraries were used to determine regions of RNA bound by proteins.
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Project description:Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP “target” RNAs. To address this, an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP, was developed. easyCLIP, provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize unamplified libraries. 213 cross-link measurements were performed and found that RBPs could be distinguished from non-RBPs by RNA cross-link rates and that target RNAs could be defined as those with a complex frequency unlikely for a random protein. easyCLIP was used to study the 33 most recurrent cancer mutations across 28 RBPs. This demonstrated increased RNA binding per RBP molecule for KHDRBS2 and A1CF cancer mutants, and showed that the PCBP1L100P/Q cancer mutation also dramatically increased RNA binding. Quantitating RBP-RNA interactions can thus categorize proteins as RBPs or not and define the impact of specific disease-associated RBP mutations on RNA binding.
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Project description:Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
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