IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080174.124

Caitlin H Lamb, Emmanuelle M Pitré, Sean Ajufo, Charlotte V Rigby, Karishma Bisht, Michael S Oade, Hamid Jalal, Cameron Myhrvold, Aartjan J W Te Velthuis

{"title":"Quantification of influenza virus mini viral RNAs using Cas13.","authors":"Caitlin H Lamb, Emmanuelle M Pitré, Sean Ajufo, Charlotte V Rigby, Karishma Bisht, Michael S Oade, Hamid Jalal, Cameron Myhrvold, Aartjan J W Te Velthuis","doi":"10.1261/rna.080174.124","DOIUrl":"10.1261/rna.080174.124","url":null,"abstract":"Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host-pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs, and it complements existing amplification-based approaches.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"126-138"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648933/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142473667","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

Widespread destabilization of Caenorhabditis elegans microRNAs by the E3 ubiquitin ligase EBAX-1. E3泛素连接酶EBAX-1广泛破坏秀丽隐杆线虫microRNA的稳定性。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080276.124

Michael W Stubna, Aditi Shukla, David P Bartel

{"title":"Widespread destabilization of Caenorhabditis elegans microRNAs by the E3 ubiquitin ligase EBAX-1.","authors":"Michael W Stubna, Aditi Shukla, David P Bartel","doi":"10.1261/rna.080276.124","DOIUrl":"10.1261/rna.080276.124","url":null,"abstract":"MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that direct mRNA repression. miRNAs are also the subject of regulation. For example, some miRNAs are destabilized through a pathway in which pairing to specialized transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. Here, we found that 22 miRNAs in Caenorhabditis elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). The impact of EBAX-1 depended on the developmental stage, with the greatest effect on the miRNA pool (14.5%) observed in L1 larvae, and the greatest number of different miRNAs affected (17) observed in germline-depleted adults. The affected miRNAs included the miR-35-42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major miRNA-destabilization pathway in the worm. The excess miR-35-42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to EBAX-1 loss had no consistent pattern of either trimming or tailing. Replacement of the 3' region of miR-43 substantially reduced EBAX-1 sensitivity, a result that differed from that observed previously for miR-35. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that a role for miRNA 3' sequences is variable in the worm.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"51-66"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648932/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142473684","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

Template switching enables chemical probing of native RNA structures. 通过模板切换，可对原生 RNA 结构进行化学探测。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.079926.123

Ian Hall, Martin O'Steen, Sophie Gold, Sarah C Keane, Chase A Weidmann

{"title":"Template switching enables chemical probing of native RNA structures.","authors":"Ian Hall, Martin O'Steen, Sophie Gold, Sarah C Keane, Chase A Weidmann","doi":"10.1261/rna.079926.123","DOIUrl":"10.1261/rna.079926.123","url":null,"abstract":"RNAs are often studied in nonnative sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods-a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added postprobing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5' and 3' ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced nonnative interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to nonnative sequence elements.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"113-125"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648929/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142507014","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

Corrigendum: Specific roles for the Ccr4-Not complex subunits in expression of the genome. 勘误：Ccr4-Not复杂亚基在基因组表达中的特殊作用。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080301.124

Nowel Azzouz, Olesya O Panasenko, Cécile Deluen, Julien Hsieh, Grégory Theiler, Martine A Collart

引用次数: 0

Investigating the role of RNA-binding protein Ssd1 in aneuploidy tolerance through network analysis. 通过网络分析研究 RNA 结合蛋白 Ssd1 在非整倍体耐受性中的作用

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080199.124

H Auguste Dutcher, Audrey P Gasch

{"title":"Investigating the role of RNA-binding protein Ssd1 in aneuploidy tolerance through network analysis.","authors":"H Auguste Dutcher, Audrey P Gasch","doi":"10.1261/rna.080199.124","DOIUrl":"10.1261/rna.080199.124","url":null,"abstract":"RNA-binding proteins (RBPs) play critical cellular roles by mediating various stages of RNA life cycles. Ssd1, an RBP with pleiotropic effects, has been implicated in aneuploidy tolerance in Saccharomyces cerevisiae but its mechanistic role remains unclear. Here, we used a network-based approach to inform on Ssd1's role in aneuploidy tolerance, by identifying and experimentally perturbing a network of RBPs that share mRNA targets with Ssd1. We identified RBPs whose bound mRNA targets significantly overlap with Ssd1 targets. For 14 identified RBPs, we then used a genetic approach to generate all combinations of genotypes for euploid and aneuploid yeast with an extra copy of chromosome XII, with and without SSD1 and/or the RBP of interest. Deletion of 10 RBPs either exacerbated or alleviated the sensitivity of wild-type and/or ssd1Δ cells to chromosome XII duplication, in several cases indicating genetic interactions with SSD1 in the context of aneuploidy. We integrated these findings with results from a global overexpression screen that identified genes whose duplication complements ssd1Δ aneuploid sensitivity. The resulting network points to a subgroup of proteins with shared roles in translational repression and P-body formation, implicating these functions in aneuploidy tolerance. Our results reveal a role for new RBPs in aneuploidy tolerance and support a model in which Ssd1 mitigates translation-related stresses in aneuploid cells.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"100-112"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648931/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142547092","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

Bioinformatics-driven refinement of the commonly used TPI nonsense-mediated decay reporter system. 生物信息学驱动的常用 TPI 无义衰变报告系统的改进

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080134.124

Laura Peter, Lara Walotka, Johannes Ptok, Caroline Meyer, Dominik Schüller, Heiner Schaal, Lisa Müller

{"title":"Bioinformatics-driven refinement of the commonly used TPI nonsense-mediated decay reporter system.","authors":"Laura Peter, Lara Walotka, Johannes Ptok, Caroline Meyer, Dominik Schüller, Heiner Schaal, Lisa Müller","doi":"10.1261/rna.080134.124","DOIUrl":"10.1261/rna.080134.124","url":null,"abstract":"The cellular nonsense-mediated decay (NMD) pathway recognizes and degrades mRNAs with unusual structural features, such as long 3' UTRs or overlapping reading frames, and therefore serves as a transcript quality control mechanism. A broad spectrum of today's knowledge about the nonsense-mediated mRNA decay pathway has been discovered using NMD reporter systems, mostly consisting of multiple exons, with a wild-type and a premature termination codon-containing variant. In a preliminary NMD study, we used the seven-exon triose phosphate isomerase (TPI) reporter and observed that in this well-known NMD reporter, surprisingly, not all splice sites are used constitutively, but additional cryptic splice sites are used. As this is more frequently observed in the construction of minigenes, especially when unknown splicing regulatory elements (SREs) are removed, for example, by shortening introns, this may affect the reliability of such reporters. To demonstrate how such minigenes can be improved in general with respect to constitutive splice site recognition, we restored an intron length in the TPI reporter or made bioinformatic adjustments to SREs or intrinsic strength of the splice sites themselves. As a result, this NMD reporter could be made more robust and specific for the evaluation of NMD sensitivity within a single transcript. The modifications of the TPI reporter shown here as examples can generally be used for the transfer of cellular multiexon transcripts to minigenes.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"32-42"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648928/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142473666","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

RNA: Reviewers for Volume 30, 2024. RNA：第 30 卷（2024 年）审稿人。

IF 4.2 3区生物学

RNA Pub Date : 2024-11-18

引用次数: 0

Beyond RNA-binding domains: determinants of protein-RNA binding. 超越 RNA 结合域：蛋白质与 RNA 结合的决定因素。

IF 4.2 3区生物学

RNA Pub Date : 2024-11-18 DOI: 10.1261/rna.080026.124

Inbal Zigdon, Miri Carmi, Sagie Brodsky, Zohar Rosenwaser, Naama Barkai, Felix Jonas

引用次数: 0

RNA: Reviewers for Volume 30, 2024. RNA：第 30 卷（2024 年）审稿人。

IF 4.2 3区生物学