IF 4.2 3区生物学

RNA Pub Date : 2024-11-18 DOI: 10.1261/rna.080282.124

Anming Huang, Lydia Riepler, Dietmar Rieder, Janine Kimpel, Alexandra Lusser

引用次数: 0

High-resolution reconstruction of a C. elegans ribosome sheds light on evolutionary dynamics and tissue specificity. 高分辨率重建秀丽隐杆线虫核糖体揭示了进化动态和组织特异性。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080103.124

Enisha Sehgal, Chloe Wohlenberg, Evan M Soukup, Marcus J Viscardi, Vitor Hugo Balasco Serrão, Joshua A Arribere

{"title":"High-resolution reconstruction of a C. elegans ribosome sheds light on evolutionary dynamics and tissue specificity.","authors":"Enisha Sehgal, Chloe Wohlenberg, Evan M Soukup, Marcus J Viscardi, Vitor Hugo Balasco Serrão, Joshua A Arribere","doi":"10.1261/rna.080103.124","DOIUrl":"10.1261/rna.080103.124","url":null,"abstract":"Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression and tissue patterning in animals. An invaluable tool in modern gene expression research is the presence of a high-resolution ribosome structure, though no such structure exists for C. elegans Here, we present a high-resolution single-particle cryogenic electron microscopy (cryo-EM) reconstruction and molecular model of a C. elegans ribosome, revealing a significantly streamlined animal ribosome. Many facets of ribosome structure are conserved in C. elegans, including overall ribosomal architecture and the mechanism of cycloheximide, whereas other facets, such as expansion segments and eL28, are rapidly evolving. We identify uL5 and uL23 as two instances of tissue-specific ribosomal protein paralog expression conserved in Caenorhabditis, suggesting that C. elegans ribosomes vary across tissues. The C. elegans ribosome structure will provide a basis for future structural, biochemical, and genetic studies of translation in this important animal system.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1513-1528"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482609/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142111518","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

A circular split nanoluciferase reporter for validating and screening putative internal ribosomal entry site elements. 用于验证和筛选推定的内部核糖体入口位点元件的环形分裂纳米荧光素酶报告器。

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080008.124

Mildred J Unti, Lisa Doetsch, Samie R Jaffrey

{"title":"A circular split nanoluciferase reporter for validating and screening putative internal ribosomal entry site elements.","authors":"Mildred J Unti, Lisa Doetsch, Samie R Jaffrey","doi":"10.1261/rna.080008.124","DOIUrl":"10.1261/rna.080008.124","url":null,"abstract":"Internal ribosomal entry sites (IRESs) recruit the ribosome to promote translation, typically in an m7G cap-independent manner. Although IRESs are well-documented in viral genomes, they have also been reported in mammalian transcriptomes, where they have been proposed to mediate cap-independent translation of mRNAs. However, subsequent studies have challenged the idea of these \"cellular\" IRESs. Current methods for screening and discovering IRES activity rely on a bicistronic reporter assay, which is prone to producing false positive signals if the putative IRES sequence has a cryptic promoter or cryptic splicing sites. Here, we report an assay for screening IRES activity using a genetically encoded circular RNA comprising a split nanoluciferase (nLuc) reporter. The circular split nLuc reporter is less susceptible to the various sources of false positives that adversely affect the bicistronic IRES reporter assay and provides a streamlined method for screening IRES activity. Using the circular split nLuc reporter, we find that nine reported cellular IRESs have minimal IRES activity. Overall, the circular split nLuc reporter offers a simplified approach for identifying and validating IRESs and exhibits reduced propensity for producing the types of false positives that can occur with the bicistronic reporter assay.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1529-1540"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482608/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141894190","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

Abolished frameshifting for predicted structure-stabilizing SARS-CoV-2 mutants: implications to alternative conformations and their statistical structural analyses. 预测的结构稳定型 SARS-CoV-2 突变体的移帧作用减弱：对替代构象及其统计结构分析的影响

IF 4.2 3区生物学

RNA Pub Date : 2024-10-16 DOI: 10.1261/rna.080035.124

Abhishek Dey, Shuting Yan, Tamar Schlick, Alain Laederach

{"title":"Abolished frameshifting for predicted structure-stabilizing SARS-CoV-2 mutants: implications to alternative conformations and their statistical structural analyses.","authors":"Abhishek Dey, Shuting Yan, Tamar Schlick, Alain Laederach","doi":"10.1261/rna.080035.124","DOIUrl":"10.1261/rna.080035.124","url":null,"abstract":"The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases, including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1437-1450"},"PeriodicalIF":4.2,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11482603/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141860802","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

Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-seq. 马拉松 RT 模板切换反应的特征和实施，以扩展 RNA-Seq 的功能。

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

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

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

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