IF 4.2 3区生物学

RNA Pub Date : 2024-08-16 DOI: 10.1261/rna.079886.123

Jadwiga Meissner, Katarzyna Eysmont, Katarzyna Matylla-Kulińska, Maria M Konarska

{"title":"Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing.","authors":"Jadwiga Meissner, Katarzyna Eysmont, Katarzyna Matylla-Kulińska, Maria M Konarska","doi":"10.1261/rna.079886.123","DOIUrl":"10.1261/rna.079886.123","url":null,"abstract":"The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1199-1212"},"PeriodicalIF":4.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11331412/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141321519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The antivirulent Staphylococcal sRNA SprC regulates CzrB efflux pump to adapt its response to Zinc toxicity 抗病毒葡萄球菌 sRNA SprC 可调控 CzrB 外排泵，使其适应锌毒性反应

IF 4.5 3区生物学

RNA Pub Date : 2024-08-01 DOI: 10.1261/rna.080122.124

Simon Raynaud, Marc Hallier, Stephane Dreano, Brice Felden, Yoann Augagneur, Helene Le Pabic

{"title":"The antivirulent Staphylococcal sRNA SprC regulates CzrB efflux pump to adapt its response to Zinc toxicity","authors":"Simon Raynaud, Marc Hallier, Stephane Dreano, Brice Felden, Yoann Augagneur, Helene Le Pabic","doi":"10.1261/rna.080122.124","DOIUrl":"https://doi.org/10.1261/rna.080122.124","url":null,"abstract":"Bacterial regulatory RNAs (sRNAs) are important players to control gene expression. In S. aureus, SprC is an antivirulent trans-acting sRNA known to base-pair with the major autolysin atl mRNA, preventing its translation. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we looked for its sRNA-RNA interactome and identified fourteen novel mRNA targets. In vitro biochemical investigations revealed that SprC binds two of them, czrB and deoD, and uses a single accessible region to regulate its targets, including Atl translation. Unlike Atl regulation, the characterization of the SprC-czrB interaction pinpointed a destabilization of czrAB co-transcript,leading to a decrease of the mRNA level that impaired CzrB Zinc efflux pump expression. On a physiological stand-point, we showed that SprC expression is detrimental to combat against Zinc toxicity. In addition, phagocyctosis assays revealed a significant, but moderate, increase of czrB mRNA level in a sprC-deleted mutant, indicating a functional link between SprC and\tczrB upon internalization in macrophages, and suggesting a role in resistance to both oxidative and Zinc burst. Altogether, our data uncover a novel pathway in which SprC is implicated, highlighting the multiple strategies employed by S. aureus to balance virulence using an RNA regulator.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"10 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141866495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Functional analysis of the zinc finger modules of the Saccharomyces cerevisiae splicing factor Luc7. 麦角菌剪接因子 Luc7 锌指模块的功能分析

IF 4.2 3区生物学

RNA Pub Date : 2024-07-16 DOI: 10.1261/rna.079956.124

Tucker J Carrocci, Samuel DeMario, Kevin He, Natalie J Zeps, Cade T Harkner, Guillaume F Chanfreau, Aaron A Hoskins

{"title":"Functional analysis of the zinc finger modules of the Saccharomyces cerevisiae splicing factor Luc7.","authors":"Tucker J Carrocci, Samuel DeMario, Kevin He, Natalie J Zeps, Cade T Harkner, Guillaume F Chanfreau, Aaron A Hoskins","doi":"10.1261/rna.079956.124","DOIUrl":"10.1261/rna.079956.124","url":null,"abstract":"Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1058-1069"},"PeriodicalIF":4.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11251517/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140892689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Transcription factors ERα and Sox2 have differing multiphasic DNA- and RNA-binding mechanisms. 转录因子 ERα 和 Sox2 具有不同的多相 DNA 和 RNA 结合机制。

IF 4.2 3区生物学

RNA Pub Date : 2024-07-16 DOI: 10.1261/rna.080027.124

Wayne O Hemphill, Halley R Steiner, Jackson R Kominsky, Deborah S Wuttke, Thomas R Cech

{"title":"Transcription factors ERα and Sox2 have differing multiphasic DNA- and RNA-binding mechanisms.","authors":"Wayne O Hemphill, Halley R Steiner, Jackson R Kominsky, Deborah S Wuttke, Thomas R Cech","doi":"10.1261/rna.080027.124","DOIUrl":"10.1261/rna.080027.124","url":null,"abstract":"Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here, we use biophysical assays to interrogate the k on, k off, and K d for DNA and RNA binding of two model human TFs, ERα and Sox2. Unexpectedly, we found that both proteins exhibit multiphasic nucleic acid-binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics can be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ERα nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, in which two apparent binding transitions are separated by a 10-20 min \"lag\" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ERα nucleic acid-binding data. Instead, simulations with a model incorporating sequential ERα monomer association, ERα nucleic acid complex isomerization, and product \"feedback\" on isomerization rate recapitulated the general kinetic trends for both ERα DNA and RNA binding. Collectively, our findings reveal that Sox2 and ERα bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ERα binding nucleic acids via a novel reaction mechanism.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1089-1105"},"PeriodicalIF":4.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11251522/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140958964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements. 镁离子缓解 mRNA 成分调控格局中的易变状态

IF 4.2 3区生物学

RNA Pub Date : 2024-07-16 DOI: 10.1261/rna.079767.123

Erdong Ding, Susmit Narayan Chaudhury, Jigneshkumar Dahyabhai Prajapati, José N Onuchic, Karissa Y Sanbonmatsu

{"title":"Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.","authors":"Erdong Ding, Susmit Narayan Chaudhury, Jigneshkumar Dahyabhai Prajapati, José N Onuchic, Karissa Y Sanbonmatsu","doi":"10.1261/rna.079767.123","DOIUrl":"10.1261/rna.079767.123","url":null,"abstract":"Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 μsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg2+ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg2+ and ligand also facilitate a more compact structure in the three-way junction region.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"992-1010"},"PeriodicalIF":4.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11251524/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

TERRA ONTseq: a long-read-based sequencing pipeline to study the human telomeric transcriptome. TERRA ONTseq：研究人类端粒转录组的基于长读数的测序管道。

IF 4.2 3区生物学

RNA Pub Date : 2024-07-16 DOI: 10.1261/rna.079906.123

Joana Rodrigues, Roberta Alfieri, Silvia Bione, Claus M Azzalin

{"title":"TERRA ONTseq: a long-read-based sequencing pipeline to study the human telomeric transcriptome.","authors":"Joana Rodrigues, Roberta Alfieri, Silvia Bione, Claus M Azzalin","doi":"10.1261/rna.079906.123","DOIUrl":"10.1261/rna.079906.123","url":null,"abstract":"The long noncoding RNA TERRA is transcribed from telomeres in virtually all eukaryotes with linear chromosomes. In humans, TERRA transcription is driven in part by promoters comprising CpG dinucleotide-rich repeats of 29 bp repeats, believed to be present in half of the subtelomeres. Thus far, TERRA expression has been analyzed mainly using molecular biology-based approaches that only generate partial and somehow biased results. Here, we present a novel experimental pipeline to study human TERRA based on long-read sequencing (TERRA ONTseq). By applying TERRA ONTseq to different cell lines, we show that the vast majority of human telomeres produce TERRA and that the cellular levels of TERRA transcripts vary according to their chromosomes of origin. Using TERRA ONTseq, we also identified regions containing TERRA transcription start sites (TSSs) in more than half of human subtelomeres. TERRA TSS regions are generally found immediately downstream from 29 bp repeat-related sequences, which appear to be more widespread than previously estimated. Finally, we isolated a novel TERRA promoter from the highly expressed subtelomere of the long arm of Chromosome 7. With the development of TERRA ONTseq, we provide a refined picture of human TERRA biogenesis and expression and we equip the scientific community with an invaluable tool for future studies.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"955-966"},"PeriodicalIF":4.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11251519/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The Catalyst: RNA and the Quest to Unlock Life's Deepest Secrets: Thomas R. Cech. Norton, New York. 2024. 催化剂：核糖核酸与解开生命深层秘密的探索：托马斯-R-切奇。纽约诺顿2024.

IF 4.5 3区生物学

RNA Pub Date : 2024-07-10 DOI: 10.1261/rna.080173.124

Thoru Pederson

引用次数: 0

Modulation of diverse biological processes by CPSF, the master regulator of mRNA 3’ ends mRNA3'末端的主调控因子CPSF对多种生物过程的调节作用

IF 4.5 3区生物学

RNA Pub Date : 2024-07-10 DOI: 10.1261/rna.080108.124

Lizhi Liu, James L. Manley

引用次数: 0

Molecular basis of human poly(A) polymerase recruitment by mPSF. mPSF 招募人类 polyA 聚合酶的分子基础。

IF 4.2 3区生物学

RNA Pub Date : 2024-06-17 DOI: 10.1261/rna.079915.123

Sofia Todesca, Felix Sandmeir, Achim Keidel, Elena Conti

{"title":"Molecular basis of human poly(A) polymerase recruitment by mPSF.","authors":"Sofia Todesca, Felix Sandmeir, Achim Keidel, Elena Conti","doi":"10.1261/rna.079915.123","DOIUrl":"10.1261/rna.079915.123","url":null,"abstract":"3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"795-806"},"PeriodicalIF":4.2,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11182016/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140306764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High-throughput quantitation of protein-RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins. 高通量量化蛋白质-RNA UV 交联效率，作为高可信度鉴定 RNA 结合蛋白的预测工具。

IF 4.2 3区生物学

RNA Pub Date : 2024-05-16 DOI: 10.1261/rna.079848.123

JohnCarlo Kristofich, Christopher V Nicchitta

{"title":"High-throughput quantitation of protein-RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins.","authors":"JohnCarlo Kristofich, Christopher V Nicchitta","doi":"10.1261/rna.079848.123","DOIUrl":"10.1261/rna.079848.123","url":null,"abstract":"UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions, however, makes attribution of RNA-binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA-binding protein (RBP) confidence scoring metric (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high- and low-confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions, for example, the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support the development of more predictive computational approaches for RBP identification.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"644-661"},"PeriodicalIF":4.2,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11098464/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139997252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

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