IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080254.124

Michael Anikin, Michael F Henry, Viktoria Hodorova, Hristo B Houbaviy, Jozef Nosek, Dimitri G Pestov, Dmitriy A Markov

{"title":"Mitochondrial mRNA and the small subunit rRNA in budding yeasts undergo 3'-end processing at conserved species-specific elements.","authors":"Michael Anikin, Michael F Henry, Viktoria Hodorova, Hristo B Houbaviy, Jozef Nosek, Dimitri G Pestov, Dmitriy A Markov","doi":"10.1261/rna.080254.124","DOIUrl":"10.1261/rna.080254.124","url":null,"abstract":"Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These include cis- and trans-regulatory factors that generate and protect the 3' ends. Here, we mapped the 3' ends of mitochondrial mRNAs of yeasts classified into multiple families of the subphylum Saccharomycotina. We found that the processing of mitochondrial 15S rRNA and mRNAs involves species-specific sequence elements, which we term 3'-end RNA processing elements (3'-RPEs). In Saccharomyces cerevisiae, the 3'-RPE has long been recognized as a conserved dodecamer sequence, which recent studies have shown specifically interacts with the nuclear genome-encoded pentatricopeptide repeat protein Rmd9. We also demonstrate that, analogous to Rmd9 in S. cerevisiae, two Rmd9 orthologs from the Debaryomycetaceae family interact with their respective 3'-RPEs found in mRNAs and 15S rRNA. Thus, Rmd9-dependent processing of mitochondrial RNA precursors may be a common mechanism among the families of the Saccharomycotina subphylum. Surprisingly, we observed that 3'-RPEs often occur upstream of stop codons in complex I subunit mRNAs from yeasts of the CUG-Ser1 clade. We examined two of these mature mRNAs and found that their stop codons are indeed removed. Thus, translation of these stop-codon-less transcripts would require a noncanonical termination mechanism. Our findings highlight Rmd9 as a key evolutionarily conserved factor in both mitochondrial mRNA metabolism and mitoribosome biogenesis in a variety of yeasts.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"208-223"},"PeriodicalIF":4.2,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11789488/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142688618","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

Microbial iCLIP2: enhanced mapping of RNA-protein interaction by promoting protein and RNA stability. 微生物iCLIP2：通过促进蛋白质和RNA的稳定性来增强RNA-蛋白相互作用的定位。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080193.124

Nina Kim Stoffel, Srimeenakshi Sankaranarayanan, Kira Müntjes, Nadine Körtel, Anke Busch, Kathi Zarnack, Julian König, Michael Feldbrügge

{"title":"Microbial iCLIP2: enhanced mapping of RNA-protein interaction by promoting protein and RNA stability.","authors":"Nina Kim Stoffel, Srimeenakshi Sankaranarayanan, Kira Müntjes, Nadine Körtel, Anke Busch, Kathi Zarnack, Julian König, Michael Feldbrügge","doi":"10.1261/rna.080193.124","DOIUrl":"10.1261/rna.080193.124","url":null,"abstract":"The entire RNA life cycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV cross-linking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP-binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus Ustilago maydis as an example. Key challenges, such as inherently high RNase and protease activity in fungi, were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of cross-link events and improved the identification of Rrm4-binding sites. Thereby, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complexes I, III, and V-revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study using U. maydis as an example might serve as a blueprint for optimizing iCLIP2 procedures in other microorganisms with high RNase/protease conditions.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"258-272"},"PeriodicalIF":4.2,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11789484/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808020","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

Rational design yields RNA-binding zinc finger domains with altered sequence specificity. 合理设计产生具有改变序列特异性的rna结合锌指结构域。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080329.124

Qishan Liang, Joy S Xiang, Gene W Yeo, Kevin D Corbett

{"title":"Rational design yields RNA-binding zinc finger domains with altered sequence specificity.","authors":"Qishan Liang, Joy S Xiang, Gene W Yeo, Kevin D Corbett","doi":"10.1261/rna.080329.124","DOIUrl":"10.1261/rna.080329.124","url":null,"abstract":"Targeting and manipulating endogenous RNAs in a sequence-specific manner is essential for both understanding RNA biology and developing RNA-targeting therapeutics. RNA-binding zinc fingers (ZnFs) are excellent candidates as designer proteins to expand the RNA-targeting toolbox, due to their compact size and modular sequence recognition. Currently, little is known about how the sequence of RNA-binding ZnF domains governs their binding site specificity. Here, we systematically introduced mutations at the RNA-contacting residues of a well-characterized RNA-binding ZnF protein, ZRANB2, and measured RNA binding of mutant ZnFs using a modified RNA bind-n-seq assay. We identified mutant ZnFs with an altered sequence specificity, preferring to bind a GGG motif instead of the GGU preferred by wild-type ZRANB2. Further, through a series of all-atom molecular dynamics simulations with ZRANB2 and RNA, we characterized changes in the hydrogen-bond network between the protein and RNA that underlie the observed sequence specificity changes. Our analysis of ZRANB2-RNA interactions both in vitro and in silico expands the understanding of ZnF-RNA recognition rules and serves as a foundation for eventual use of RNA-binding ZnFs for programmable RNA targeting.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"150-163"},"PeriodicalIF":4.2,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11789483/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808024","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

DRBD18 acts as a transcript-specific RNA editing auxiliary factor in Trypanosoma brucei. DRBD18在布鲁氏锥虫中作为转录特异性RNA编辑辅助因子。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080295.124

Parul Pandey, Katherine Wackowski, Ashutosh P Dubey, Laurie K Read

{"title":"DRBD18 acts as a transcript-specific RNA editing auxiliary factor in Trypanosoma brucei.","authors":"Parul Pandey, Katherine Wackowski, Ashutosh P Dubey, Laurie K Read","doi":"10.1261/rna.080295.124","DOIUrl":"10.1261/rna.080295.124","url":null,"abstract":"Uridine insertion/deletion (U-indel) RNA editing of mitochondrial transcripts is a posttranscriptional modification in kinetoplastid organisms, resulting in the generation of mature mRNAs from cryptic precursors. This RNA editing process involves a multiprotein complex holoenzyme and multiple accessory factors. Recent investigations have highlighted the pivotal involvement of accessory RNA-binding proteins (RBPs) in modulating RNA editing in Trypanosoma brucei, often in a transcript-specific manner. DRBD18 is a multifunctional RBP that reportedly impacts the stability, processing, export, and translation of nuclear-encoded mRNAs. However, mass spectrometry studies report DRBD18-RESC interactions, prompting us to investigate its role in mitochondrial U-indel RNA editing. In this study, we demonstrate the specific and RNase-sensitive interaction of DRBD18 with multiple RESC factors. Depletion of DRBD18 through RNA interference in procyclic form T. brucei leads to a significant reduction in the levels of edited A6 and COIII mitochondrial transcripts, whereas its overexpression causes a notable increase in the abundance of these edited mRNAs. RNA immunoprecipitation/qRT-PCR analysis indicates a direct role for DRBD18 in A6 and COIII mRNA editing. We also examined the impact of arginine methylation of DRBD18 in the editing process, revealing that the hypomethylated form of DRBD18, rather than the arginine-methylated version, is essential for promoting these editing events. In conclusion, our findings demonstrate that DRBD18 directly affects the editing of A6 and COIII mRNAs, with its function being modulated by its arginine methylation status, marking the first report of a mitochondrial function for this protein and identifying it as a newly characterized RNA editing auxiliary factor.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"245-257"},"PeriodicalIF":4.2,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11789491/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142807964","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 precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing. 有序双模板接力cDNA文库制备RNA测序的精度、灵敏度和适应性提高。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080318.124

Lucas Ferguson, Heather E Upton, Sydney C Pimentel, Chris Jeans, Nicholas T Ingolia, Kathleen Collins

{"title":"Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing.","authors":"Lucas Ferguson, Heather E Upton, Sydney C Pimentel, Chris Jeans, Nicholas T Ingolia, Kathleen Collins","doi":"10.1261/rna.080318.124","DOIUrl":"10.1261/rna.080318.124","url":null,"abstract":"Sequencing RNAs that are biologically processed or degraded to less than ∼100 nt typically involves multistep, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced ordered two-template relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced nonproductive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 pg (<1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or posttranscriptional modifications.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"224-244"},"PeriodicalIF":4.2,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11789487/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142772146","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

Two dynamic N-terminal regions are required for function in ribosomal RNA adenine dimethylase family members. 核糖体 RNA 腺嘌呤二甲基化酶家族成员的功能需要两个动态的 N 端区域。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080068.124

Danielle A McGaha, Alexandrea Collins, Luqman O Ajisafe, Calvin C Perdigao, Jordan L Bondrowski, Karen Fetsch, Jack A Dunkle

{"title":"Two dynamic N-terminal regions are required for function in ribosomal RNA adenine dimethylase family members.","authors":"Danielle A McGaha, Alexandrea Collins, Luqman O Ajisafe, Calvin C Perdigao, Jordan L Bondrowski, Karen Fetsch, Jack A Dunkle","doi":"10.1261/rna.080068.124","DOIUrl":"10.1261/rna.080068.124","url":null,"abstract":"Prominent members of the ribosomal RNA adenine dimethylase (RRAD) family of enzymes facilitate ribosome maturation by dimethylating 2 nt of small subunit rRNA, including the human DIMT1 and bacterial KsgA enzymes. A subgroup of RRAD enzymes, named erythromycin resistance methyltransferases (Erm), dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays, we found that the N-terminal region is critical for activity in RRAD enzymes: The N-terminal basic region promotes rRNA binding, and the conserved motif likely assists in juxtaposing the adenosine substrate and the S-adenosylmethionine cofactor. Our results and emerging structural data suggest that this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding, forming a cap on the active site required for methylation.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"164-180"},"PeriodicalIF":4.2,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11789486/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142627183","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

Protein binding in an mRNA 5'-UTR sterically hinders translation. mRNA 5'-UTR中的蛋白质结合在空间上阻碍了翻译。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-22 DOI: 10.1261/rna.080136.124

Simon Felder, Irma M Nelson, Breanne M Hatfield, Kevin M Weeks

引用次数: 0

Meeting report ASOBIOTICS 2024: an interdisciplinary symposium on antisense-based programmable RNA antibiotics. ASOBIOTICS 2024：基于反义的可编程RNA抗生素的跨学科研讨会。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-15 DOI: 10.1261/rna.080347.124

Jorg Vogel, Franziska Faber, Lars Barquist, Anke Sparmann, Linda Popella, Chandradhish Ghosh

{"title":"Meeting report ASOBIOTICS 2024: an interdisciplinary symposium on antisense-based programmable RNA antibiotics.","authors":"Jorg Vogel, Franziska Faber, Lars Barquist, Anke Sparmann, Linda Popella, Chandradhish Ghosh","doi":"10.1261/rna.080347.124","DOIUrl":"https://doi.org/10.1261/rna.080347.124","url":null,"abstract":"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 Wurzburg, Germany, on September 12-13th, 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.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

eIF4F-mediated Dysregulation of mRNA Translation in Cancer. 肿瘤中eif4f介导的mRNA翻译失调。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-14 DOI: 10.1261/rna.080340.124

Mehdi Amiri, Niaz Mahmood, Soroush Tahmasebi, Nahum Sonenberg

引用次数: 0

Cytoplasmic Regulation of the Poly(A) Tail Length as a Potential Therapeutic Target. Poly(A)尾长度的细胞质调控作为潜在的治疗靶点。

IF 4.2 3区生物学

RNA Pub Date : 2025-01-13 DOI: 10.1261/rna.080333.124

Mercedes Fernandez, Raul Mendez

{"title":"Cytoplasmic Regulation of the Poly(A) Tail Length as a Potential Therapeutic Target.","authors":"Mercedes Fernandez, Raul Mendez","doi":"10.1261/rna.080333.124","DOIUrl":"https://doi.org/10.1261/rna.080333.124","url":null,"abstract":"Virtually all mRNAs acquire a poly(A) tail co-transcriptionally, but its length is dynamically regulated in the cytoplasm in a transcript-specific manner. The length of the poly(A) tail plays a crucial role in determining mRNA translation, stability, and localization. This dynamic regulation of poly(A) tail length is widely used to create post-transcriptional gene expression programs, allowing for precise temporal and spatial control. Dysregulation of poly(A) tail length has been linked to various diseases, including cancers, inflammatory and cardiovascular disorders, and neurological syndromes. Cytoplasmic poly(A) tail length is maintained by a dynamic equilibrium between cis-acting elements and cognate factors that promote deadenylation or polyadenylation, enabling rapid gene expression reprogramming in response to internal and external cellular cues. While cytoplasmic deadenylation and its pathophysiological implications have been extensively studied, cytoplasmic polyadenylation and its therapeutic potential remain less explored. This review discusses the distribution, regulation, and mechanisms of Cytoplasmic Polyadenylation Element-Binding Proteins (CPEBs), highlighting their dual roles in either promoting or repressing gene expression depending on cellular context. We also explore their involvement in diseases such as tumor progression and metastasis, along with their potential as targets for novel therapeutic strategies.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979899","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

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