IF 4.2 3区生物学

RNA Pub Date : 2024-09-16 DOI: 10.1261/rna.080156.124

Malithi I Jayasinghe, Krishna J Patel, Jane E Jackman

{"title":"Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis.","authors":"Malithi I Jayasinghe, Krishna J Patel, Jane E Jackman","doi":"10.1261/rna.080156.124","DOIUrl":"10.1261/rna.080156.124","url":null,"abstract":"Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1315-1327"},"PeriodicalIF":4.2,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11404450/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141601427","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

Increasing MicroRNA Abundance by Targeting Biogenesis from the Primary Transcript with Steric-Blocking Antisense Oligonucleotides 利用立体阻断反义寡核苷酸靶向初级转录本的生物生成，提高 MicroRNA 的丰度

IF 4.5 3区生物学

RNA Pub Date : 2024-09-10 DOI: 10.1261/rna.080021.124

Mallory A Havens, Anthony J Hinrich, Frank Rigo, Michelle L Hastings

{"title":"Increasing MicroRNA Abundance by Targeting Biogenesis from the Primary Transcript with Steric-Blocking Antisense Oligonucleotides","authors":"Mallory A Havens, Anthony J Hinrich, Frank Rigo, Michelle L Hastings","doi":"10.1261/rna.080021.124","DOIUrl":"https://doi.org/10.1261/rna.080021.124","url":null,"abstract":"MicroRNAs (miRNAs) are regulators of gene expression, and their dysregulation is linked to cancer and other diseases, making them important therapeutic targets. Several strategies for targeting and modulating miRNA activity are being explored. For example, steric blocking antisense oligonucleotides (ASOs) can reduce miRNA activity by either blocking binding sites on specific mRNAs or base-pairing to the miRNA itself to prevent its interaction with the target mRNAs. ASOs have been less explored as a tool to elevate miRNA levels, which could also be beneficial for treating disease. In this study, using the PKD1/miR-1225 gene locus as an example, where miR-1225 is located within a PKD1 intron, we demonstrate an ASO-based strategy that increases miRNA abundance by enhancing biogenesis from the primary miRNA transcript. Disruptions in PKD1 and miR-1225 are associated with autosomal dominant polycystic kidney disease (ADPKD) and various cancers, respectively, making them important therapeutic targets. We investigated PKD1 sequence variants reported in ADPKD that are located within the sequence shared by miR-1225 and PKD1, and identified one that causes a reduction in miR-1225 without affecting PKD1. We show that this reduction in miR-1225 can be recovered by treatment with a steric-blocking ASO. The ASO-induced increase in miR-1225 correlates with a decrease in the abundance of predicted miR-1225 cellular mRNA targets. This study demonstrates that miRNA abundance can be elevated using ASOs targeted to the primary transcript. This steric-blocking ASO-based approach has broad potential application as a therapeutic strategy for diseases that could be treated by modulating miRNA biogenesis.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"169 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142196076","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

tRNAVal allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei 在干酪乳杆菌中，tRNAVal 可与位于摆动位置的未修饰尿苷进行四向解码

IF 4.5 3区生物学

RNA Pub Date : 2024-09-10 DOI: 10.1261/rna.080155.124

Riko Sugita, Vincent Guérineau, David Touboul, Satoko Yoshizawa, Kazuyuki Takai, Chie Tomikawa

{"title":"tRNAVal allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei","authors":"Riko Sugita, Vincent Guérineau, David Touboul, Satoko Yoshizawa, Kazuyuki Takai, Chie Tomikawa","doi":"10.1261/rna.080155.124","DOIUrl":"https://doi.org/10.1261/rna.080155.124","url":null,"abstract":"Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson-Crick basepairs. In bacterial tRNA, 5-hydroxyuridine (ho5U) derivatives xo5U [x: methyl (mo5U), carboxymethyl (cmo5U), and methoxycarbonylmethyl (mcmo5U)] present at the wobble positions of tRNAs are responsible for recognition of NYN codon families. These modifications of U34 allow basepairing not only with A and G but also with U and in some cases C. mo5U was originally found in Gram-positive bacteria, and cmo5U and mcmo5U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei, Gram-positive bacteria and lactic acid bacteria, lacks the modification enzyme genes for xo5U biosynthesis. Nevertheless, L. casei has only one type of tRNAVal with the anticodon UAC [tRNAVal(UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNAUnd) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNAVal is unmodified and that there is no tRNAUnd expression in the cells. In addition, in vitro transcribed tRNAUnd was not aminoacylated by L. casei valyl-tRNA synthetase suggesting that tRNAUnd is not able to accept valine, even if expressed in cells. Correspondingly, native tRNAVal(UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNAVal decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"42 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142225103","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

Regulatory Interplay Between SR Proteins Governs CLK1 Kinase Splice Variants Production SR蛋白之间的调控相互作用控制着CLK1激酶剪接变体的产生

IF 4.5 3区生物学

RNA Pub Date : 2024-09-09 DOI: 10.1261/rna.080107.124

Lulzim Shkreta, Aurelie Delannoy, Johanne Toutant, Benoit Chabot

{"title":"Regulatory Interplay Between SR Proteins Governs CLK1 Kinase Splice Variants Production","authors":"Lulzim Shkreta, Aurelie Delannoy, Johanne Toutant, Benoit Chabot","doi":"10.1261/rna.080107.124","DOIUrl":"https://doi.org/10.1261/rna.080107.124","url":null,"abstract":"The CLK1 kinase phosphorylates SR proteins to modulate their splicing regulatory activity. Skipping of alternative exon 4 on the CLK1 pre-mRNA produces a CLK1 variant lacking the catalytic site. Here, we aimed to understand how various SR proteins integrate into the regulatory program that controls CLK1 exon 4 splicing. Previously, we observed that the depletion of SRSF10 promoted the inclusion of CLK1 exon 4. Using expression of tagged proteins and CRISPR/Cas9-mediated knockouts in HCT116 cells, we now identify TRA2b, TRA2a, SRSF4, SRSF5, SRSF7, SRSF8 and SRSF9 as activators of exon 4 inclusion. In contrast, SRSF3, SRSF10 and SRSF12 elicit exon 4 skipping. Using CRISPR/dCas13Rx and RNA immunoprecipitation assays, we map an enhancer in exon 4 interacting with TRA2b. Notably, CLK1 kinase inhibitors antagonized the repressor activity of HA-SRSF10, HA-SRSF12 and HA-SRSF3. Our results suggest that CLK1 exon 4 inclusion is determined primarily by a balance between the activities of TRA2 proteins and CLK-phosphorylated SRSF3. CLK-phosphorylated SRSF10 and SRSF12 would interact with TRA2 proteins to prevent their enhancer activity, allowing SRSF3 to enforce exon 4 skipping more efficiently. Our study provides insight into the complex regulatory network controlling the alternative splicing of CLK1, which uses CLK1-mediated phosphorylation of SR proteins to regulate the inclusion of catalytic exon 4 in CLK1 transcripts.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"26 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142196077","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

Corrigendum: Gene regulation by a glycine riboswitch singlet uses a finely tuned energetic landscape for helical switching 更正：甘氨酸核糖开关单体利用微调的能量景观进行螺旋切换的基因调控

IF 4.5 3区生物学

RNA Pub Date : 2024-09-01 DOI: 10.1261/rna.080151.124

Chad D. Torgerson, David A. Hiller, Shira Stav, Scott A. Strobel

引用次数: 0

Corrigendum: Translation regulation by a guanidine-II riboswitch is highly tunable in sensitivity, dynamic range, and apparent cooperativity 更正：鸟苷-II 核糖开关的翻译调控在灵敏度、动态范围和明显的合作性方面具有高度可调性

IF 4.5 3区生物学

RNA Pub Date : 2024-09-01 DOI: 10.1261/rna.080150.124

Caroline M. Focht, David A. Hiller, Sabrina G. Grunseich, Scott A. Strobel

引用次数: 0

Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription. 腺苷修饰阻碍了 SARS-CoV-2 RNA 依赖性 RNA 转录。

IF 4.2 3区生物学

RNA Pub Date : 2024-08-16 DOI: 10.1261/rna.079991.124

Laura R Snyder, Ingrid Kilde, Artem Nemudryi, Blake Wiedenheft, Markos Koutmos, Kristin S Koutmou

{"title":"Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription.","authors":"Laura R Snyder, Ingrid Kilde, Artem Nemudryi, Blake Wiedenheft, Markos Koutmos, Kristin S Koutmou","doi":"10.1261/rna.079991.124","DOIUrl":"10.1261/rna.079991.124","url":null,"abstract":"SARS-CoV-2, the causative virus of the COVID-19 pandemic, follows SARS and MERS as recent zoonotic coronaviruses causing severe respiratory illness and death in humans. The recurrent impact of zoonotic coronaviruses demands a better understanding of their fundamental molecular biochemistry. Nucleoside modifications, which modulate many steps of the RNA life cycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or antiviral effect is unknown. Regardless, the viral RNA-dependent RNA polymerase will encounter these modifications as it transcribes through the viral genomic RNA. We investigated the functional consequences of nucleoside modification on the pre-steady state kinetics of SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted transcription system with modified RNA templates. Our findings show that N 6-methyladenosine and 2'-O-methyladenosine modifications slow the rate of viral transcription at magnitudes specific to each modification, which has the potential to impact SARS-CoV-2 genome maintenance.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1141-1150"},"PeriodicalIF":4.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11331411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141470603","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

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein. RNA 模板 5' 端的结构和序列会影响 R2 逆转录质子蛋白插入转基因。

IF 4.2 3区生物学

RNA Pub Date : 2024-08-16 DOI: 10.1261/rna.080031.124

Sarah M Palm, Connor A Horton, Xiaozhu Zhang, Kathleen Collins

{"title":"Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.","authors":"Sarah M Palm, Connor A Horton, Xiaozhu Zhang, Kathleen Collins","doi":"10.1261/rna.080031.124","DOIUrl":"10.1261/rna.080031.124","url":null,"abstract":"R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1227-1245"},"PeriodicalIF":4.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11331408/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498880","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

Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning. 对 RNA 聚合酶和逆转录酶保真度的离散测量揭示了进化调整。

IF 4.2 3区生物学

RNA Pub Date : 2024-08-16 DOI: 10.1261/rna.080002.124

Vladimir Potapov, Stanislas Krudup, Sean Maguire, Irem Unlu, Shengxi Guan, Jackson A Buss, Benedict A Smail, Trevor van Eeuwen, Martin S Taylor, Kathleen H Burns, Jennifer L Ong, Robert J Trachman

{"title":"Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.","authors":"Vladimir Potapov, Stanislas Krudup, Sean Maguire, Irem Unlu, Shengxi Guan, Jackson A Buss, Benedict A Smail, Trevor van Eeuwen, Martin S Taylor, Kathleen H Burns, Jennifer L Ong, Robert J Trachman","doi":"10.1261/rna.080002.124","DOIUrl":"10.1261/rna.080002.124","url":null,"abstract":"Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1246-1258"},"PeriodicalIF":4.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11331410/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141470604","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

Loss of ADAR1 protein induces changes in small RNA landscape in hepatocytes. ADAR1 蛋白的缺失会诱导肝细胞中的小 RNA 结构发生变化。

IF 4.2 3区生物学

RNA Pub Date : 2024-08-16 DOI: 10.1261/rna.080097.124

Kristina Roučová, Václav Vopálenský, Tomáš Mašek, Edgar Del Llano, Jan Provazník, Jonathan J M Landry, Nayara Azevedo, Edvard Ehler, Vladimír Beneš, Martin Pospíšek

{"title":"Loss of ADAR1 protein induces changes in small RNA landscape in hepatocytes.","authors":"Kristina Roučová, Václav Vopálenský, Tomáš Mašek, Edgar Del Llano, Jan Provazník, Jonathan J M Landry, Nayara Azevedo, Edvard Ehler, Vladimír Beneš, Martin Pospíšek","doi":"10.1261/rna.080097.124","DOIUrl":"10.1261/rna.080097.124","url":null,"abstract":"In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis.","PeriodicalId":21401,"journal":{"name":"RNA","volume":" ","pages":"1164-1183"},"PeriodicalIF":4.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11331409/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141284673","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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