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

An internal loop region is responsible for inherent target specificity of bacterial cold-shock proteins. 细菌冷休克蛋白的固有目标特异性是由一个内环区域造成的。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080163.124

Satoshi Hasegawa, Rerina Inose, Mizuki Igarashi, Megumi Tsurumaki, Motofumi Saito, Tatsuo Yanagisawa, Akio Kanai, Teppei Morita

{"title":"An internal loop region is responsible for inherent target specificity of bacterial cold-shock proteins.","authors":"Satoshi Hasegawa, Rerina Inose, Mizuki Igarashi, Megumi Tsurumaki, Motofumi Saito, Tatsuo Yanagisawa, Akio Kanai, Teppei Morita","doi":"10.1261/rna.080163.124","DOIUrl":"10.1261/rna.080163.124","url":null,"abstract":"Cold-shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold-shock domain (CSD) that contains RNA-binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here, we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD, and the N-terminal portion with Lys4 of CspA, are important for determining their target specificities. Pull-down assays suggest that these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit a different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD-type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding the functional specialization of Csps.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"67-85"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648934/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142473665","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

Development of bioconjugate-based delivery systems for nucleic acids. 开发基于生物共轭物的核酸输送系统。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080273.124

Aniket Wahane, Vishal Kasina, Mounika Pathuri, Ciara Marro-Wilson, Anisha Gupta, Frank J Slack, Raman Bahal

{"title":"Development of bioconjugate-based delivery systems for nucleic acids.","authors":"Aniket Wahane, Vishal Kasina, Mounika Pathuri, Ciara Marro-Wilson, Anisha Gupta, Frank J Slack, Raman Bahal","doi":"10.1261/rna.080273.124","DOIUrl":"10.1261/rna.080273.124","url":null,"abstract":"Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1-13"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648935/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142547091","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 oligonucleotides containing N7-regioisomer of guanosine: influence on thermodynamic properties and structure of RNA duplexes. 含有鸟苷 N7-regioisomer 的寡核苷酸。对 RNA 双链体热力学性质和结构的影响。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080106.124

Aleksandra Jarmolowicz, Nivedita Dutta, Witold Andralojc, Joanna Sarzynska, Grzegorz Framski, Daniel Baranowski, Jerzy Boryski, Ansuman Lahiri, Zofia Gdaniec, Elzbieta Kierzek, Ryszard Kierzek

{"title":"The oligonucleotides containing N7-regioisomer of guanosine: influence on thermodynamic properties and structure of RNA duplexes.","authors":"Aleksandra Jarmolowicz, Nivedita Dutta, Witold Andralojc, Joanna Sarzynska, Grzegorz Framski, Daniel Baranowski, Jerzy Boryski, Ansuman Lahiri, Zofia Gdaniec, Elzbieta Kierzek, Ryszard Kierzek","doi":"10.1261/rna.080106.124","DOIUrl":"10.1261/rna.080106.124","url":null,"abstract":"During the chemical synthesis of the purine riboside, N7-regioisomer is kinetically formed, whereas N9-regioisomer is a thermodynamically formed product. We have studied the effect of substituting N9-regioisomer of guanosine with its N7-regioisomer (N7-guanosine, 7G) at a central position of several RNA duplexes. We found that this single substitution by 7G severely diminished their thermodynamic stabilities when 7G paired with C and U, but remarkably, led to a significant amount of stabilization in most of the duplexes when forming mismatches with G and A. The extent of stabilization was observed to be dependent on the sequence and orientation of neighboring base pairs of N7-guanosine. 1D and 2D NMR studies on the duplexes along with extensive molecular dynamics simulations revealed the conformational differences occurring due to the substitution of G by 7G, and it was observed that the thermodynamic results were largely explainable by considering the formation of stable noncanonical hydrogen bonding interactions, although other interactions such as stacking and electrostatic interactions could also play a role. These observations can have important applications in the design of RNA-based disease diagnostics and therapeutics.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"86-99"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142522880","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

Conserved role for spliceosomal component PRPF40A in microexon splicing. 剪接体成分 PRPF40A 在微外显子剪接中的保守作用

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080142.124

Bikash Choudhary, Adam Norris

引用次数: 0

RNA fold prediction by Monte Carlo in graph space and the statistical mechanics of tertiary interactions. 通过蒙特卡罗图空间和三级相互作用的统计力学预测 RNA 折叠。

IF 4.2 3区生物学

RNA Pub Date : 2024-12-16 DOI: 10.1261/rna.080216.124

Ethan N H Phan, Chi H Mak

{"title":"RNA fold prediction by Monte Carlo in graph space and the statistical mechanics of tertiary interactions.","authors":"Ethan N H Phan, Chi H Mak","doi":"10.1261/rna.080216.124","DOIUrl":"10.1261/rna.080216.124","url":null,"abstract":"Using a graph representation of RNA structures, we have studied the ensembles of secondary and tertiary graphs of two sets of RNA with Monte Carlo simulations. The first consisted of 91 target ribozyme and riboswitch sequences of moderate lengths (<150 nt) having a variety of secondary, H-type pseudoknots and kissing loop interactions. The second set consisted of 71 more diverse sequences across many RNA families. Using a simple empirical energy model for tertiary interactions and only sequence information for each target as input, the simulations examined how tertiary interactions impact the statistical mechanics of the fold ensembles. The results show that the graphs proliferate enormously when tertiary interactions are possible, producing an entropic driving force for the ensemble to access folds having tertiary structures even though they are overall energetically unfavorable in the energy model. For each of the targets in the two test sets, we assessed the quality of the model and the simulations by examining how well the simulated structures were able to predict the native fold, and compared the results to fold predictions from ViennaRNA. Our model generated good or excellent predictions in a large majority of the targets. Overall, this method was able to produce predictions of comparable quality to Vienna, but it outperformed Vienna for structures with H-type pseudoknots. The results suggest that while tertiary interactions are predicated on real-space contacts, their impacts on the folded structure of RNA can be captured by graph space information for sequences of moderate lengths, using a simple tertiary energy model for the loops, the base pairs, and base stacks.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"14-31"},"PeriodicalIF":4.2,"publicationDate":"2024-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11648927/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142507013","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最新文献