RNA最新文献

{"title":"The PAZ domain of <i>Aedes aegypti</i> Dicer 2 is critical for accurate and high-fidelity size determination of virus-derived small interfering RNAs.","authors":"Melinda Reuter, Rhys H Parry, Melanie McFarlane, Rommel J Gestuveo, Rozeena Arif, Alexander A Khromykh, Benjamin Brennan, Margus Varjak, Alfredo Castello, Lars Redecke, Esther Schnettler, Alain Kohl","doi":"10.1261/rna.080149.124","DOIUrl":"10.1261/rna.080149.124","url":null,"abstract":"<p><p>The exogenous siRNA (exo-siRNA) pathway is a critical RNA interference response involved in controlling arbovirus replication in mosquito cells. It is initiated by the detection of viral long double-stranded RNA (dsRNA) by the RNase III enzyme Dicer 2 (Dcr2), which is processed into predominantly 21 nt virus-derived small interfering RNAs (vsiRNAs) that are taken up by the Argonaute 2 (Ago2) protein to target viral single-stranded RNAs. The detailed understanding of Dicer structure, function and domains owes much to studies outside the context of viral infection and studies in model organisms, and as such how Dcr2 domains contribute to detecting viral dsRNA to mount antiviral responses in infected mosquito cells remains less well understood. Here, we used a Dcr2 reconstitution system in <i>Aedes aegypti</i> derived Dcr2 knockout (KO) cells to assess the contribution of the PIWI-Argonaute-Zwille (PAZ) domain to induction of the exo-siRNA pathway following infection with Semliki Forest virus (SFV; <i>Togaviridae</i>, <i>Alphavirus</i>). Amino acids critical for PAZ activity were identified, and loss of PAZ function affected the production of 21 nt vsiRNAs-with enrichment of 22 nt SFV-derived small RNAs observed-and silencing activity. This study establishes PAZ domain's functional contribution to Dcr2 processing of viral dsRNA to 21 nt vsiRNAs.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"679-691"},"PeriodicalIF":4.2,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12001973/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143415090","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}

{"title":"ASOBIOTICS 2024: an interdisciplinary symposium on antisense-based programmable RNA antibiotics.","authors":"Jörg Vogel, Franziska Faber, Lars Barquist, Anke Sparmann, Linda Popella, Chandradhish Ghosh","doi":"10.1261/rna.080347.124","DOIUrl":"10.1261/rna.080347.124","url":null,"abstract":"<p><p>The international symposium ASOBIOTICS 2024 brought together scientists across disciplines to discuss the challenges of advancing antibacterial antisense oligomers (ASOs) from basic research to clinical application. Hosted by the Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg, Germany, on September 12-13, 2024, the event featured presentations covering major milestones and current challenges of this antimicrobial technology and its applications against pathogens, commensals, and bacterial viruses. General design principles and modification of ASOs based on peptide nucleic acid (PNA) or phosphorodiamidate-morpholino-oligomer (PMO) chemistry, promising cellular RNA targets, new delivery technologies, as well as putative resistance mechanisms, were discussed. A panel discussion noted the challenge of nomenclature: antibacterial ASOs lack a single, universally used name. To address this, the term \"asobiotics\" was proposed to unite a community of like-minded scientists that are committed to advancing ASOs as antimicrobials. A consistent name will simplify literature searches and help scientists and funders appreciate the potential of programmable RNA antibiotics to combat antimicrobial resistance and enable precise microbiome editing.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"465-474"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912906/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010994","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}

{"title":"An incredible life in science: Joseph G. Gall (1928-2024).","authors":"Svetlana Deryusheva, Ji-Long Liu, Zehra F Nizami, Gaëlle J S Talross, Susan A Gerbi","doi":"10.1261/rna.080406.125","DOIUrl":"10.1261/rna.080406.125","url":null,"abstract":"","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"453-464"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12046345/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370284","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}

{"title":"Global analysis by LC-MS/MS of <i>N6</i>-methyladenosine and inosine in mRNA reveal complex incidence.","authors":"Stanislav Stejskal, Veronika Rájecká, Helena Covelo-Molares, Ketty Sinigaglia, Květoslava Brožinová, Linda Kašiarová, Michaela Dohnálková, Paul Eduardo Reyes-Gutierrez, Hana Cahová, Liam P Keegan, Mary A O'Connell, Štěpánka Vaňáčová","doi":"10.1261/rna.080324.124","DOIUrl":"10.1261/rna.080324.124","url":null,"abstract":"<p><p>The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m⁶A, m⁶A_m, inosine, and m¹A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m⁶A_m marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m⁶A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"514-528"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912911/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922795","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}

{"title":"Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit.","authors":"Sepideh Fakhretaha Aval, Amal Seffouh, Kyung-Mee Moon, Leonard J Foster, Joaquin Ortega, Kurt Fredrick","doi":"10.1261/rna.080335.124","DOIUrl":"10.1261/rna.080335.124","url":null,"abstract":"<p><p>The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, form contacts with the SRL that are critical for GTP hydrolysis and factor function. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in <i>Escherichia coli</i> Isolated ΔSRL particles were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We find that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this activity is blocked in the absence of the SRL.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"585-599"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912913/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143060505","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}

{"title":"Thermodynamic control of mismatch discrimination for extensive splicing regulation of PKM pre-mRNA.","authors":"Natalia Bartyś, Jolanta Lisowiec-Wąchnicka, Anna Pasternak","doi":"10.1261/rna.080212.124","DOIUrl":"10.1261/rna.080212.124","url":null,"abstract":"<p><p>In this article, we present an approach to maximizing the splicing regulatory properties of splice-switching oligonucleotide (SSO) designed to regulate alternative splicing of pyruvate kinase M (<i>PKM)</i> pre-mRNA. The studied SSO interacts with the regulatory element in exon 10 of <i>PKM</i> pre-mRNA and contributes to a significant reduction of PKM2 level with a simultaneous increase of the PKM1 isoform. This SSO forms a duplex not only with the regulatory fragment of exon 10 but also with a similar RNA fragment of intron 9. The impact of this unspecific interaction on SSO regulatory properties, as well as the functional role of the intron 9 fragment, remains debatable. Herein, two types of modified nucleotides, unlocked nucleic acids (UNAs) and locked nucleic acids (LNAs), were used to study mismatch discrimination within duplexes involving modified SSOs. Our studies showed that LNAs increased mismatch discrimination, resulting in stronger regulatory properties of SSO. On the other hand, UNA reduced mismatch discrimination, decreasing the potentially therapeutic properties of SSO. The results indicate that specific interactions with exon 10 are more favorable for therapeutic applications than simultaneous hybridization with intron 9 and exon 10. The results also suggest the lack of a regulatory role for the intron 9 target site in alternative splicing of PKM pre-mRNA.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"475-485"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912903/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142966496","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}

{"title":"Live-cell imaging of circular and long noncoding RNAs associated with FUS pathological aggregates by Pepper fluorescent RNA.","authors":"Erika Vitiello, Francesco Castagnetti, Lorenzo Stufera Mecarelli, Eleonora D'Ambra, Paolo Tollis, Giancarlo Ruocco, Pietro Laneve, Elisa Caffarelli, Davide Mariani, Irene Bozzoni","doi":"10.1261/rna.080119.124","DOIUrl":"10.1261/rna.080119.124","url":null,"abstract":"<p><p>Lately, important advancements in visualizing RNAs in fixed and live cells have been achieved. Although mRNA imaging techniques are well-established, the development of effective methods for studying noncoding RNAs (ncRNAs) in living cells is still challenging but necessary, as they show a variety of functions and intracellular localizations, including participation in highly dynamic processes like phase transition, which is still poorly studied in vivo. Addressing this issue, we tagged two exemplary ncRNAs with the fluorescent RNA (fRNA) Pepper. Specifically, we showed that circ-HDGFRP3 interacts with p-bodies and is recruited in pathological FUS aggregates in a dynamic fashion, and we super-resolved its distribution in such condensates via structured illumination microscopy. Moreover, we tracked the long noncoding RNA (lncRNA) nHOTAIRM1, a motor neuron-specific constituent of stress granules, monitoring its behavior throughout the oxidative-stress response in physiological and pathological conditions. Overall, as fRNA development progresses, our work demonstrates an effective use of Pepper for monitoring complex processes, such as phase transition, in living cells through the visualization of circular RNAs (circRNAs) and lncRNAs with super-resolution power.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"529-548"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912908/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142954236","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}

{"title":"<i>Schizosaccharomyces pombe pus1</i> mutants are temperature sensitive due to decay of tRNA^Ile(UAU) by the 5'-3' exonuclease Dhp1, primarily targeting the unspliced pre-tRNA.","authors":"Franziska Stegemann, Erin Marcus, Savanah Neupert, Sarah Ostrowski, David H Mathews, Eric M Phizicky","doi":"10.1261/rna.080315.124","DOIUrl":"10.1261/rna.080315.124","url":null,"abstract":"<p><p>The pseudouridylase Pus1 catalyzes pseudouridine (Ψ) formation at multiple uridine residues in tRNAs, and in some snRNAs and mRNAs. Although Pus1 is highly conserved, and mutations are associated with human disease, little is known about eukaryotic Pus1 biology. Here, we show that <i>Schizosaccharomyces pombe pus1</i>Δ mutants are temperature sensitive due to decay of tRNA^Ile(UAU), as tRNA^Ile(UAU) levels are reduced, and its overexpression suppresses the defect. We show that tRNA^Ile(UAU) is degraded by the 5'-3' exonuclease Dhp1 (ortholog of <i>Saccharomyces cerevisiae</i> Rat1), as each of four spontaneous <i>pus1</i>Δ suppressors had <i>dhp1</i> mutations and restored tRNA^Ile(UAU) levels, and two suppressors that also restored tRNA^Ile(UAU) levels had mutations in <i>tol1</i> (<i>S. cerevisiae MET22</i> ortholog), predicted to inhibit Dhp1. We show that Pus1 modifies U₂₇, U₃₄, and U₃₆ of tRNA^Ile(UAU), raising the question about how these modifications prevent decay. Our results suggest that Dhp1 targets unspliced pre-tRNA^Ile(UAU), as a <i>pus1</i>Δ strain in which the only copy of tRNA^Ile(UAU) has no intron [<i>tI(UAU)-i</i>Δ] is temperature resistant and undergoes no detectable decay, and the corresponding <i>pus1</i>Δ <i>tI(UAU)-WT</i> strain accumulates unspliced pre-tRNA^Ile(UAU) Moreover, the predicted exon-intron structure of pre-tRNA^Ile(UAU) differs from the canonical bulge-helix-loop structure compatible with tRNA splicing, and a <i>pus1</i>Δ <i>tI(UAU)i-var</i> strain with intron mutations predicted to improve exon-intron structure is temperature resistant and undergoes little decay. These results suggest that decay of tRNA^Ile(UAU) by Dhp1 in <i>pus1</i>Δ strains occurs at the level of unspliced pre-tRNA^Ile(UAU), implying a substantial role for one or more of the Ψ residues in stabilizing the pre-tRNA structure for splicing.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"566-584"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912914/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143029578","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}

{"title":"rRFtargetDB: a database of Ago1-mediated targets of ribosomal RNA fragments.","authors":"Lingyu Guan, Sathyanarayanan Vaidhyanathan, Andrey Grigoriev","doi":"10.1261/rna.080285.124","DOIUrl":"10.1261/rna.080285.124","url":null,"abstract":"<p><p>rRNA-derived fragments (rRFs) are a class of emerging posttranscriptional regulators of gene expression likely binding to the transcripts of target genes. However, the lack of knowledge about such targets hinders our understanding of rRF functions or binding mechanisms. The paucity of resources supporting the identification of the targets of rRFs creates a bottleneck in the fast-developing field. We have previously analyzed chimeric reads in cross-linked Argonaute1-RNA complexes to help infer the guide-target pairs and binding mechanisms of multiple rRFs based on experimental data in human HEK293 cells. To efficiently disseminate these results to the research community, we designed a web-based database rRFtargetDB that preserves most of the experimental results after the removal of noise and has a user-friendly interface with flexible query options and filters allowing users to obtain comprehensive information on rRFs (or targets) of interest. rRFtargetDB is populated by ∼163,000 experimentally determined unique rRF-mRNA pairs (∼60,000 supported by ≥2 reads). Almost 30,000 rRF isoforms produced >385,000 (>156,000 with ≥2 reads) chimeras with all types of RNA targets (mRNAs and noncoding RNAs). Further analyses suggested hypothetical modes of interactions, supported by secondary structures of potential guide-target hybrids and binding motifs, essential for understanding the targeting mechanisms of rRFs. All these results (ranging from the weakest to the strongest experimental support) are presented in rRFtargetDB, whose goal is to provide a resource for building users' hypotheses on the potential roles of rRFs for experimental validation. Further, we illustrate the value/application of the database in several examples.rRFtargetDB is freely accessible at https://grigoriev-lab.camden.rutgers.edu/tardb.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"486-496"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912905/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142954240","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}

{"title":"New reporters for monitoring cellular NMD.","authors":"Hanna Alalam, Monika Šafhauzer, Per Sunnerhagen","doi":"10.1261/rna.080272.124","DOIUrl":"10.1261/rna.080272.124","url":null,"abstract":"<p><p>Nonsense-mediated decay (NMD) is a eukaryotic surveillance pathway that controls degradation of cytoplasmic transcripts with aberrant features. NMD-controlled RNA degradation acts to regulate a large fraction of the mRNA population. It has been implicated in cellular responses to infections and environmental stress, as well as in deregulation of tumor-promoting genes. NMD is executed by a set of three core factors conserved in evolution, UPF1-3, as well as additional influencing proteins such as kinases. Monitoring NMD activity is challenging due to the difficulties in quantitating RNA decay rates in vivo, and consequently, it has also been problematic to identify new factors influencing NMD. Here, we developed a genetic selection system in yeast to capture new components affecting NMD status. The reporter constructs link NMD target sequences with nutrient-selectable genetic markers. By crossing these reporters into a genome-wide library of deletion mutants and quantitating colony growth on a selective medium, we robustly detect previously known NMD components in a high-throughput fashion. In addition, we identify novel mutations influencing NMD status and implicate ribosome recycling as important for NMD. By using our constructed combinations of promoters, NMD target sequences, and selectable markers, the system can also efficiently detect mutations with a minor effect, or in special environments. Furthermore, it can be used to explore how NMD acts on targets of different structures.</p>","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"600-611"},"PeriodicalIF":4.2,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11912909/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143067906","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}