Similar Datasets
Project description:The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and lncRNA function. How Dbp2p functions in these seemingly unrelated biological roles is not known. An important step towards addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using denaturing tandem affinity purification followed by UV-crosslinking. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and most markedly to non-coding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially interacts with sites near the 3’ ends. These sites closely correspond to regions where RNA-DNA hybrids (R-loop) form, and to binding sites of the RNA helicase Sen1p, a component of the Nrd1-Nab3-Sen1 (NNS) complex, which functions in transcription termination and 3' processing of non-coding RNAs in yeast. We also show that Dbp2p interacts in an RNA-independent manner with Sen1p in vivo. We further observe Dbp2p crosslinks to tRNAs and other RNAs also at sites where RNA-loops form. Collectively, our data link Dbp2p to diverse non-coding RNAs, to Sen1p, and to R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for seemingly unrelated cellular roles of Dbp2p.
2018-04-22 | GSE113488 | GEO
Project description:The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and lncRNA function. How Dbp2p functions in these seemingly unrelated biological roles is not known. An important step towards addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using denaturing tandem affinity purification followed by UV-crosslinking. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and most markedly to non-coding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially interacts with sites near the 3’ ends. These sites closely correspond to regions where RNA-DNA hybrids (R-loop) form, and to binding sites of the RNA helicase Sen1p, a component of the Nrd1-Nab3-Sen1 (NNS) complex, which functions in transcription termination and 3' processing of non-coding RNAs in yeast. We also show that Dbp2p interacts in an RNA-independent manner with Sen1p in vivo. We further observe Dbp2p crosslinks to tRNAs and other RNAs also at sites where RNA-loops form. Collectively, our data link Dbp2p to diverse non-coding RNAs, to Sen1p, and to R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for seemingly unrelated cellular roles of Dbp2p.
2018-04-22 | GSE113487 | GEO
Project description:The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and long noncoding RNA (lncRNA) function. It is not understood how Dbp2p performs these seemingly unrelated biological roles. An important step toward addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using UV-crosslinking, denaturing tandem affinity purification, and next generation sequencing. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and markedly to noncoding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially crosslinks at sites near the 3' ends. These sites coincide with regions where RNA-DNA hybrids (R-loops) form and with binding sites of Sen1p, another RNA helicase that functions in transcription termination and 3' processing of noncoding RNAs. We show that Dbp2p interacts in an RNA-independent manner with Sen1p in vivo. Dbp2p crosslinks to tRNAs and other RNAs also at sites where R-loops form. Collectively, our data link Dbp2p to noncoding RNAs, Sen1p, and R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for diverse cellular roles of Dbp2p.
| S-EPMC6239179 | biostudies-literature
Project description:The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops
| PRJNA451258 | ENA
Project description:The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops [iCLIP]
| PRJNA451261 | ENA
Project description:The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops [RNA-Seq]
| PRJNA451260 | ENA
Project description:R loops are three-stranded nucleic acid structures consisting of an RNA:DNA heteroduplex and a "looped-out" nontemplate strand. As aberrant formation and persistence of R loops block transcription elongation and cause DNA damage, mechanisms that resolve R loops are essential for genome stability. Here we show that the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase DDX21 efficiently unwinds R loops and that depletion of DDX21 leads to accumulation of cellular R loops and DNA damage. Significantly, the activity of DDX21 is regulated by acetylation. Acetylation by CBP inhibits DDX21 activity, while deacetylation by SIRT7 augments helicase activity and overcomes R-loop-mediated stalling of RNA polymerases. Knockdown of SIRT7 leads to the same phenotype as depletion of DDX21 (i.e., increased formation of R loops and DNA double-strand breaks), indicating that SIRT7 and DDX21 cooperate to prevent R-loop accumulation, thus safeguarding genome integrity. Moreover, DDX21 resolves estrogen-induced R loops on estrogen-responsive genes in breast cancer cells, which prevents the blocking of transcription elongation on these genes.
| S-EPMC5580657 | biostudies-literature
Project description:The present study demonstrates how TOP3B is involved in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/DNA hybrid IP-western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-mass spectrometry and IP-western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with senataxin. We propose a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage.
| S-EPMC10575568 | biostudies-literature
Project description:The non-structural proteins (nsPs) of the chikungunya virus (CHIKV) form the virus's replication complex. They are known to participate in several functions that allow efficient replication of the virus in diverse host systems. One such function is evading the host defense system such as RNA interference (RNAi). Two nsPs of CHIKV, namely, nsP2 and nsP3, were found to suppress the host/vector RNAi machinery and exhibit RNAi suppressor activity. The present study was undertaken to identify interacting partners of CHIKV-nsP3 in Aedes aegypti. We performed pull-down assays with the mass spectrometry approach and showed the interaction of CHIKV-nsP3 with several Aedes proteins. Further co-immunoprecipitation assays revealed that CHIKV-nsP3 interacts with RM62F, a DEAD-box containing RNA known to play roles in multiple gene regulatory processes such as alternative splicing, RNA release, and also is a component of Ago2-RISC complex.Supplementary informationThe online version contains supplementary material available at 10.1007/s13337-021-00734-y.
| S-EPMC8630140 | biostudies-literature
Project description:Structured RNAs and RNA-protein complexes (RNPs) fold through complex pathways that are replete with misfolded traps, and many RNAs and RNPs undergo extensive conformational changes during their functional cycles. These folding steps and conformational transitions are frequently promoted by RNA chaperone proteins, notably by superfamily 2 (SF2) RNA helicase proteins. The two largest families of SF2 helicases, DEAD-box and DEAH-box proteins, share evolutionarily conserved helicase cores, but unwind RNA helices through distinct mechanisms. Recent studies have advanced our understanding of how their distinct mechanisms enable DEAD-box proteins to disrupt RNA base pairs on the surfaces of structured RNAs and RNPs, while some DEAH-box proteins are adept at disrupting base pairs in the interior of RNPs. Proteins from these families use these mechanisms to chaperone folding and promote rearrangements of structured RNAs and RNPs, including the spliceosome, and may use related mechanisms to maintain cellular messenger RNAs in unfolded or partially unfolded conformations.
| S-EPMC5960804 | biostudies-literature