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RNA-biotin based pull down assay with Hela Nuclear Extract followed by RNA-seq

ABSTRACT: We aimed to discover trans-acting RNA molecules involved in mRNA 3’ processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3’ processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3’ processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used polyA site (PAS) RNA substrates, SV40 late (SVL), and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3’ end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3’ processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. We extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE98218 | GEO | 2017/07/01

SECONDARY ACCESSION(S): PRJNA384324

REPOSITORIES: GEO

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RNA-biotin based pull down assay with polyA site (PAS) RNA substrates in Hela Nuclear Extract followed by RNA-seq to discover trans-acting RNA molecules involved in mRNA 3 processing

Project description:We aimed to discover trans-acting RNA molecules involved in mRNA 3 processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3 processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3 processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used two polyA site (PAS) RNA substrates, SV40 late (SVL) and adenovirus L3 pre-mRNAs, and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3 end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3 processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. we extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing. See related experiments: E-MTAB-5384; E-MTAB-5389

2017-06-01 | E-MTAB-5383 | biostudies-arrayexpress

RNA MS2-tag based affinity method was used to purify 3' processing factors associated with intronic PAS processing

Project description:three copies of the MS2 hairpin were fused to the intronic PAS of the nr3c1 gene. To make a negative control RNA substrate, the core hexamer AAUAAA within PAS was mutated to AACAAA. Subsequently, the RNA substrate was incubated with HeLa NE, and the mRNA 3' processing complex was purified using an MS2 tag

2023-02-27 | PXD040405 | iProX

Fip1 iCLIP-seq in Hela cells without treatment or treated with negative control anti-sense oligos (ASO) or snoRD50a ASO to study how snoRD50a affects Fip1/RNA interaction in mRNA 3' processing

Project description:We aimed to discover trans-acting RNA molecules involved in mRNA 3 processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3 processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3 processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Fip1 protein is an essential mRNA 3' processing factor. Our in vitro data suggested that snoRD50a affects Fip1/RNA interaction in SV40 late (SVL) polyA site 3' processing. To determine whether snoRD50a influences Fip1/RNA interaction at transcriptomic level in vitro, we performed Fip1 iCLIP-seq experiments in Hela cells transfected with control NC ASO (negative control anti-sense DNA) or snoRD50a ASO.

2017-06-01 | E-MTAB-5384 | biostudies-arrayexpress

Identification of human mRNA processing complexes bound to histone H2A.2 3’ untranslated region

Project description:Processing of 3’ untranslated region (3’-UTR) of precursor messenger RNAs (pre-mRNA) is a fundamental step in mRNA maturation, where the mRNA transcript is cleaved at a specific site, located downstream the open reading frame of the encoded gene. The position of the cleavage site is determined by cis-acting RNA sequence elements, which are recognized with high specificity by large 3’ end processing multiprotein complexes. Two main categories of metazoan 3’ pre-mRNA processing have been identified so far, describing elaboration of either canonical mRNAs – occurring during all phases of the cell cycle and containing a characteristic Poly-Adenylation Signal (PAS) element – or histone mRNAs – prevalently occurring during the S phase and containing a Histone Downstream Element (HDE). In this work, we isolate both human endogenous canonical and histone 3’ pre-mRNA processing complexes from human nuclear extracts by using a pre-mRNA containing the 3’UTR sequence of replication-dependent histone H2A.2. This H2A.2 pre-mRNA (H2A_4m), containing both an HDE sequence and a PAS element in a native overlaid position, was modified with a 5’ photocleavable biotin tag, and with modification of few bases to allow complex assembly and purification by affinity chromatography on streptavidin-agarose beads, elution by UV irradiation and subsequent mass spectrometric identification of bound 3’ mRNA processing factors.

2022-12-09 | PXD027636 | Pride

Identification of mRNA processing complexes bound to histone H2A.2 3’ untranslated region

2022-12-09 | PXD034984 | Pride

Excessive MiR-365b-5p Maturation via N6-methyladenosine Regulates Chondrocyte Apoptosis and Phenotype by NCL/MiR-365b-5p/FHL1 Axis in Osteoarthritis

Project description:RNA pull-down assays were performed as the protocol of the PureBinding RNA-Protein pull-down Kit (Geneseed, Guangzhou, China). Specific biotinylated probes that hybridize to target pri-miR-365 were obtained from Ribobio. The probes (200pmol) targeting pri-miR-365 or control probes and RNA samples of chondrocytes were added to the pull-down system. After the mixture of RNA and probes was denaturated at 85°C for 5 minutes, and hybridized at room temperature for 2 hours, 100 µl streptavidin-coated magnetic beads were added to the mixture. Non-specifically bound RNAs were removed by hybridization, capture, and washing. The proteins that interacted with pri-miR-365 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, pri-miR-365-interacting bands were subjected to mass spectrometry (Beijing Genomics Institute, Shenzhen, China). The result was gained to establish a proteomic library.

2022-11-21 | PXD038257 | iProX

A heterochromatin specific RNA export pathway facilitates piRNA production

Project description:PIWI-interacting RNAs (piRNAs), a class of small RNAs that guide transposon silencing in animals, are processed in the cytoplasm from RNA Polymerase II transcripts. How piRNA precursors, which often lack RNA maturation signatures and thereby violate quality control checkpoints, achieve nuclear export is unknown. Here, we uncover a germline-specific RNA export pathway in Drosophila, that escorts piRNA precursors from their heterochromatic origins to nuage, the cytoplasmic piRNA processing centres. This pathway connects canonical nuclear export factors—the RNA helicase UAP56, the NXF cofactor Nxt1/p15, and the exportin Crm1/Xpo1—with Nxf3, a variant of the mRNA exporter Nxf1/Tap. Nxf3 recruitment to nascent piRNA precursors occurs via the heterochromatin protein 1-variant Rhino and CG13741/Bootlegger, a new piRNA pathway factor, thereby making piRNA precursor export independent of RNA processing events. Thus, similar to retroviral hijacking of cellular export factors, piRNA precursor export evolved to bend canonical gene expression rules through bypassing nuclear RNA surveillance mechanisms.

2019-03-29 | PXD012717 | Pride

microRNA-seq in circCDYL probe and NC probe

Project description:CircRNA pull-down was performed as previously reported. Briefly, SK-BR-3 was fixed with 1% formaldehyde and lysed by co-IP buffer and the cluster was sonicated. CircCDYL-specific and NC biotinylated probes were added to mixture tobind circCDYL. Next, C1 streptavidin magnetic beads was added to pull down circCDYL and circCDYL-binding RNA. Finally, total RNA was extracted from the magnetic beads and followed by qRT-PCR detection of circCDYL and miR-92b-3p.

2021-05-18 | GSE174541 | GEO

Protein interactome of small RNA fragments: identification and functional implications

Project description:Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules (such as miRNAs or siRNAs), also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interaction networks of seven RFs originating from tRNA, snoRNA and snRNA. In addition, we performed an in silico screen of the selected RFs against publicly available cross-linking and immunoprecipitation datasets. We determined that the RF interactome comprises a large number of proteins, which were generally different from those that interact with their parental full length RNAs. Proteins that were differentially bound by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carbocyclic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in emerging RNA-protein regulatory networks.

2018-10-24 | PXD010075 | Pride

The fission yeast MTREC complex targets CUTs and unspliced mRNAs to the nuclear exosome

Project description:Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. However, the mechanism by which they are recognized and targeted to the exosome is not fully understood. Here, we report that the MTREC complex, which has recently been shown to promote degradation of meiotic mRNAs and regulatory ncRNAs, is also the major nuclear exosome targeting complex for CUTs and unspliced pre-mRNAs in Schizosaccharomyces pombe. MTREC complex specifically binds to CUTs, meiotic mRNAs and unspliced pre-mRNA transcripts and targets these RNAs for degradation by the nuclear exosome, while the TRAMP complex has only a minor role in this process. The MTREC complex physically interacts with the nuclear exosome and with various RNA-binding and -processing complexes, coupling RNA processing to the RNA degradation machinery. Our study reveals the central role of the evolutionarily conserved MTREC complex in RNA quality control, and in the recognition and elimination of CUTs.

2015-04-22 | PXD001908 | Pride

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