IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080032.124

Li-Tao Guo, Anastasiya Grinko, Sara Olson, Alexander M Leipold, Brenton Graveley, Antoine-Emmanuel Saliba, Anna Marie Pyle

{"title":"Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-seq.","authors":"Li-Tao Guo, Anastasiya Grinko, Sara Olson, Alexander M Leipold, Brenton Graveley, Antoine-Emmanuel Saliba, Anna Marie Pyle","doi":"10.1261/rna.080032.124","DOIUrl":"10.1261/rna.080032.124","url":null,"abstract":"End-to-end RNA-sequencing methods that capture 5'-sequence content without cumbersome library manipulations are of great interest, particularly for analysis of long RNAs. While template-switching methods have been developed for RNA sequencing by distributive short-read RTs, such as the MMLV RTs used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those derived from group II introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multistep template-switching reaction carried out by RTs, in this case, by MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized the nucleotide specificity of nontemplated addition (NTA) reaction that occurs when the RT extends past the RNA 5'-terminus. We then evaluated the binding specificity of specialized template-switching oligonucleotides, optimizing their sequences and chemical properties to guide efficient template-switching reaction. Having dissected and optimized these individual steps, we then unified them into a procedure for performing RNA sequencing with MarathonRT enzymes, using a well-characterized RNA reference set. The resulting reads span a six-log range in transcript concentration and accurately represent the input RNA identities in both length and composition. We also performed RNA-seq from total human RNA and poly(A)-enriched RNA, with short- and long-read sequencing demonstrating that MarathonRT enhances the discovery of unseen RNA molecules by conventional RT. Altogether, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1495-1512"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482623/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036803","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 small noncoding RNA Vaultrc5 is dispensable to mouse development. 小非编码 RNA Vaultrc5 在小鼠发育过程中是不可或缺的。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080161.124

Mahendra Prajapat, Laura Sala, Joana A Vidigal

{"title":"The small noncoding RNA Vaultrc5 is dispensable to mouse development.","authors":"Mahendra Prajapat, Laura Sala, Joana A Vidigal","doi":"10.1261/rna.080161.124","DOIUrl":"10.1261/rna.080161.124","url":null,"abstract":"Vault RNAs (vtRNAs) are evolutionarily conserved small noncoding RNAs transcribed by RNA polymerase III. Vault RNAs were initially described as components of the vault particle, but have since been assigned multiple vault-independent functions, including regulation of PKR activity, apoptosis, autophagy, lysosome biogenesis, and viral particle trafficking. The full-length transcript has also been described as a noncanonical source of miRNAs, which are processed in a DICER-dependent manner. As central molecules in vault-dependent and independent processes, vtRNAs have been attributed numerous biological roles, including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss-of-function allele. Because Vaultrc5 is the sole murine vtRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts, pointing to a potential role for vtRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1465-1476"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482604/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142111519","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

Branch site recognition by the spliceosome. 剪接体对分支位点的识别。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080198.124

Jonas Tholen

引用次数: 0

lncRNA BC200 is processed into a stable Alu monomer. lncRNA BC200 被加工成稳定的 Alu 单体。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080152.124

Evan P Booy, Daniel Gussakovsky, Mira Brown, Rowan Shwaluk, Mark W Nachtigal, Sean A McKenna

{"title":"lncRNA BC200 is processed into a stable Alu monomer.","authors":"Evan P Booy, Daniel Gussakovsky, Mira Brown, Rowan Shwaluk, Mark W Nachtigal, Sean A McKenna","doi":"10.1261/rna.080152.124","DOIUrl":"10.1261/rna.080152.124","url":null,"abstract":"The noncoding RNA BC200 is elevated in human cancers and is implicated in translation regulation as well as cell survival and proliferation. Upon BC200 overexpression, we observed correlated expression of a second, smaller RNA species. This RNA is expressed endogenously and exhibits cell-type-dependent variability relative to BC200. Aptamer-tagged expression constructs confirmed that the RNA is a truncated form of BC200, and sequencing revealed a modal length of 120 nt; thus, we refer to the RNA fragment as BC120. We present a methodology for accurate and specific detection of BC120 and establish that BC120 is expressed in several normal human tissues and is also elevated in ovarian cancer. BC120 exhibits remarkable stability relative to BC200 and is resistant to knockdown strategies that target the 3' unique sequence of BC200. Combined knockdown of BC200 and BC120 exhibits greater phenotypic impacts than knockdown of BC200 alone, and overexpression of BC120 negatively impacts translation of a GFP reporter, providing insight into a potential translational regulatory role for this RNA. The presence of a novel, truncated, and stable form of BC200 adds complexity to the investigation of this noncoding RNA that must be considered in future studies of BC200 and other related Alu RNAs.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1477-1494"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482611/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142047140","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

Improved functions for nonlinear sequence comparison using SEEKR. 使用 SEEKR 进行非线性序列比较的改进功能。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080188.124

Shuang Li, Quinn E Eberhard, Luke Ni, J Mauro Calabrese

引用次数: 0

DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43. DNAJA2 和 Hero11 介导了 TDP-43 类似的构象扩展和聚集抑制。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080165.124

Andy Y W Lam, Kotaro Tsuboyama, Hisashi Tadakuma, Yukihide Tomari

{"title":"DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43.","authors":"Andy Y W Lam, Kotaro Tsuboyama, Hisashi Tadakuma, Yukihide Tomari","doi":"10.1261/rna.080165.124","DOIUrl":"10.1261/rna.080165.124","url":null,"abstract":"Many RNA-binding proteins (RBPs) contain low-complexity domains (LCDs) with prion-like compositions. These long intrinsically disordered regions regulate their solubility, contributing to their physiological roles in RNA processing and organization. However, this also makes these RBPs prone to pathological misfolding and aggregation that are characteristic of neurodegenerative diseases. For example, TAR DNA-binding protein 43 (TDP-43) forms pathological aggregates associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that highly disordered, hydrophilic, and charged heat-resistant obscure (Hero) proteins may have similar effects. Specifically, Hero proteins can maintain the activity of other proteins from denaturing conditions in vitro, while their overexpression can suppress cellular aggregation and toxicity associated with aggregation-prone proteins. However, it is unclear how these protective effects are achieved. Here, we used single-molecule FRET to monitor the conformations of the aggregation-prone prion-like LCD of TDP-43. While we observed high conformational heterogeneity in wild-type LCD, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, an Hsp40 chaperone, DNAJA2, and a Hero protein, Hero11, stabilized extended states of the LCD, consistent with their ability to suppress the aggregation of TDP-43. Our results link single-molecule effects on conformation to macro effects on bulk aggregation, where a Hero protein, like a chaperone, can maintain the conformational integrity of a client protein to prevent its aggregation.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1422-1436"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482610/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141907525","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

Translation elongation inhibitors stabilize select short-lived transcripts 翻译延伸抑制剂可稳定某些短寿命转录本

IF 4.5 3区生物学

RNA Pub Date : 2024-09-18 DOI: 10.1261/rna.080138.124

Nicolle A. Rosa-Mercado, Allen R. Buskirk, Rachel Green

引用次数: 0

Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea. 鉴定所有细菌和古细菌门中核糖体前体 RNA 的领导-拖曳螺旋。

IF 4.2 3区生物学

RNA Pub Date : 2024-09-16 DOI: 10.1261/rna.080091.124

Bryan T Gemler, Benjamin R Warner, Ralf Bundschuh, Kurt Fredrick

{"title":"Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea.","authors":"Bryan T Gemler, Benjamin R Warner, Ralf Bundschuh, Kurt Fredrick","doi":"10.1261/rna.080091.124","DOIUrl":"10.1261/rna.080091.124","url":null,"abstract":"Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli, complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1264-1276"},"PeriodicalIF":4.2,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11404451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141752613","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

NMDtxDB: data-driven identification and annotation of human NMD target transcripts. NMDtxDB：人类 NMD 目标转录本的数据驱动识别和注释。

IF 4.2 3区生物学

RNA Pub Date : 2024-09-16 DOI: 10.1261/rna.080066.124

Thiago Britto-Borges, Niels H Gehring, Volker Boehm, Christoph Dieterich

{"title":"NMDtxDB: data-driven identification and annotation of human NMD target transcripts.","authors":"Thiago Britto-Borges, Niels H Gehring, Volker Boehm, Christoph Dieterich","doi":"10.1261/rna.080066.124","DOIUrl":"10.1261/rna.080066.124","url":null,"abstract":"The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use generally established rules. We present a data set with four cell lines and combinations for SMG5, SMG6, and SMG7 knockdowns or SMG7 knockout. Based on this data set, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information (CDS) from Ensembl, Gencode consensus Ribo-seq ORFs, and OpenProt to enhance the CDS annotation of novel transcript isoforms. In summary, 302,889 transcripts were obtained from the transcriptome assembly process, out of which 24% are absent from Ensembl database annotations, 48,213 contain a premature stop codon, and 6433 are significantly upregulated in three or more comparisons of NMD active versus deficient cell lines. We present an in-depth view of these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1277-1291"},"PeriodicalIF":4.2,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11404449/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879337","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

IF 4.2 3区生物学

RNA Pub Date : 2024-09-16 DOI: 10.1261/rna.080011.124

Jingping Geng, Magdalena Chrabaszczewska, Karol Kurpiejewski, Anna Stankiewicz-Drogon, Marzena Jankowska-Anyszka, Edward Darzynkiewicz, Renata Grzela

{"title":"Cap-related modifications of RNA regulate binding to IFIT proteins.","authors":"Jingping Geng, Magdalena Chrabaszczewska, Karol Kurpiejewski, Anna Stankiewicz-Drogon, Marzena Jankowska-Anyszka, Edward Darzynkiewicz, Renata Grzela","doi":"10.1261/rna.080011.124","DOIUrl":"10.1261/rna.080011.124","url":null,"abstract":"All cells in our body are equipped with receptors to recognize pathogens and trigger a rapid defense response. As a result, foreign molecules are blocked, and cells are alerted to the danger. Among the many molecules produced in response to viral infection are interferon-induced proteins with tetratricopeptide repeats (IFITs). Their role is to recognize foreign mRNA and eliminate it from the translational pool of transcripts. In the present study, we used biophysical methods to characterize the interactions between the IFIT1 protein and its partners IFIT2 and IFIT3. IFIT1 interacts with IFIT3 with nanomolar binding affinity, which did not change significantly in the presence of the preformed IFIT2/3 complex. The interactions between IFIT2 and IFIT3 and IFIT1 and IFIT2 were one order of magnitude weaker. We also present kinetic data of the interactions between the IFIT protein complex and short RNA bearing various modifications at the 5' end. We show kinetic parameters for interaction between the IFIT complex and RNA with m6Am modification. The results show that the cap-adjacent m6Am modification is a stronger signature than cap1 alone. It blocks the formation of a complex between IFIT proteins and m7Gpppm6Am-RNA much more effectively than other cap modifications. In contrast, m6A in the 5'UTR is not recognized by IFIT proteins and does not contribute to translation repression by IFIT proteins. The data obtained are important for understanding the regulation of expression of genetic information. They indicate that 2'-O and m6Am modifications modulate the availability of mRNA molecules for proteins of innate immune response.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1292-1305"},"PeriodicalIF":4.2,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11404448/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141620891","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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