IF 4.5 3区生物学

RNA Pub Date : 2024-04-16 DOI: 10.1261/rna.079813.123

Rebecca J. Haugen, Catherine Barnier, Nathan D. Elrod, Hua Luo, Madeline K. Jensen, Ping Ji, Craig A. Smibert, Howard D. Lipshitz, Eric J. Wagner, P. Lydia Freddolino, Aaron C. Goldstrohm

{"title":"Regulation of the Drosophila transcriptome by Pumilio and the CCR4-NOT deadenylase complex","authors":"Rebecca J. Haugen, Catherine Barnier, Nathan D. Elrod, Hua Luo, Madeline K. Jensen, Ping Ji, Craig A. Smibert, Howard D. Lipshitz, Eric J. Wagner, P. Lydia Freddolino, Aaron C. Goldstrohm","doi":"10.1261/rna.079813.123","DOIUrl":"https://doi.org/10.1261/rna.079813.123","url":null,"abstract":"The sequence-specific RNA-binding protein Pumilio controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells in culture. We also use an improved RNA co-immunoprecipitation method to identify Pumilio-bound mRNAs in Drosophila embryos. Integration of these datasets with the locations of Pumilio binding motifs across the transcriptome reveal novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observe concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling reveals that Pumilio binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pumilio-mediated repression are not influenced by their content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"75 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140603408","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

mRNA epitranscriptomics. 简介：mRNA 表转录组学。

IF 4.5 3区生物学

RNA Pub Date : 2024-04-16 DOI: 10.1261/rna.079993.124

Kate D Meyer, Tao Pan

引用次数: 0

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2) 线粒体 TCA 循环酶苹果酸脱氢酶（MDH2）的细胞膜 RNA 结合

IF 4.5 3区生物学

RNA Pub Date : 2024-04-12 DOI: 10.1261/rna.079925.123

Michelle Noble, Aindrila Chatterjee, Thileepan Sekaran, Thomas Schwarzl, Matthias W Hentze

{"title":"Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2)","authors":"Michelle Noble, Aindrila Chatterjee, Thileepan Sekaran, Thomas Schwarzl, Matthias W Hentze","doi":"10.1261/rna.079925.123","DOIUrl":"https://doi.org/10.1261/rna.079925.123","url":null,"abstract":"Several enzymes of intermediary metabolism have been identified to bind RNA in 2 cells, with potential consequences for the bound RNAs and/or the enzyme. In this 3 study, we investigate the RNA-binding activity of the mitochondrial enzyme malate 4 dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and 5 the malate-aspartate shuttle. We confirmed in cellulo RNA-binding of MDH2 using 6 orthogonal biochemical assays and performed enhanced crosslinking and 7 immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous 8 MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, 9 although the latter are abundant in the milieu of the mature protein. Subcellular 10 fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions 11 occur predominantly outside of mitochondria. We also found that a cytosolically-12 retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that 13 mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA 14 binding increased when cellular NAD+ levels (MDH2’s co-factor) was 15 pharmacologically diminished, suggesting that the metabolic state of cells affects RNA 16 binding. Taken together, our data implicate an as yet unidentified function of MDH2 17 binding RNA in the cytosol.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"10 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140602662","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

The novel pre-rRNA detection workflow “Riboprobing” allows simple identification of undescribed RNA species. 新颖的前 RNA 检测工作流程 "Riboprobing "可简单识别未描述的 RNA 物种。

IF 4.5 3区生物学

RNA Pub Date : 2024-04-05 DOI: 10.1261/rna.079912.123

Magdalena Gerhalter, Lisa Kofler, Gertrude Zisser, Juliane Merl-Pham, Stefanie M. Hauck, Helmut Bergler

{"title":"The novel pre-rRNA detection workflow “Riboprobing” allows simple identification of undescribed RNA species.","authors":"Magdalena Gerhalter, Lisa Kofler, Gertrude Zisser, Juliane Merl-Pham, Stefanie M. Hauck, Helmut Bergler","doi":"10.1261/rna.079912.123","DOIUrl":"https://doi.org/10.1261/rna.079912.123","url":null,"abstract":"Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, Northern Blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with Primer Extension, both approaches often use radioactivity and are time consuming and costly. Here we present “Riboprobing” a reverse transcription-based workflow extended by linker ligation for easy and fast detection and characterization of various pre-rRNA species and their 5` as well as 3` ends. Using standard molecular biology lab equipment, our technique allows reliable discrimination of pre-rRNA species not resolved by Northern Blotting (e.g.: 27SA2, 27SA3 and 27SB). The method can be successfully used for analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA, arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that is lacking most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5’ETS for the assembly process.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"95 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140589465","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

Inefficient recruitment of DDX39B impedes pre-spliceosome assembly on FOXP3 introns DDX39B 的低效招募阻碍了 FOXP3 内含子上的前剪接体组装

IF 4.5 3区生物学

RNA Pub Date : 2024-04-04 DOI: 10.1261/rna.079933.123

Chloe K Nagasawa, Aaron O Bailey, William Russell, Mariano A. Garcia-Blanco

{"title":"Inefficient recruitment of DDX39B impedes pre-spliceosome assembly on FOXP3 introns","authors":"Chloe K Nagasawa, Aaron O Bailey, William Russell, Mariano A. Garcia-Blanco","doi":"10.1261/rna.079933.123","DOIUrl":"https://doi.org/10.1261/rna.079933.123","url":null,"abstract":"Forkhead box P3 (FOXP3) is the master fate-determining transcription factor in regulatory T (Treg) cells and is essential for their development, function and homeostasis. Mutations in FOXP3 cause immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, and aberrant expression of FOXP3 has been implicated in other diseases such as multiple sclerosis and cancer. We previously demonstrated that pre-mRNA splicing of FOXP3 RNAs is highly sen-sitive to levels of DExD-box polypeptide 39B (DDX39B) and here we investigate the mechanism of this sensitivity. FOXP3 introns have cytidine (C)-rich/uridine (U)-poor polypyrimidine (py) tracts that are responsible for their inefficient splicing and confer sensitivity to DDX39B. We show that there is a deficiency in the assembly of commitment complexes (CCs) on FOXP3 introns, which is consistent with the lower affinity of U2AF2 for C-rich/U-poor py tracts. Our data indicate an even stronger effect on the conversion of CCs to pre-spliceosomes. We propose that this is due to an altered conformation that U2AF2 adopts when it binds to C-rich/U-poor py tracts and that this conformation has a lower affinity for DDX39B. As a consequence, CCs assembled on FOXP3 introns are defective in recruiting DDX39B and this leads to inefficient assembly of pre-spliceosome complexes.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"36 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140589468","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

Computational analysis of super-resolved in situ sequencing data reveals genes modified by immune-tumor contact events 对超分辨原位测序数据的计算分析揭示了免疫肿瘤接触事件改变的基因

IF 4.5 3区生物学

RNA Pub Date : 2024-04-04 DOI: 10.1261/rna.079801.123

Michal Danino-Levi, Tal Goldberg, Maya Keter, Nikol Akselrod, Noa Shprach-Buaron, Modi Safra, Gonen Singer, Shahar Alon

{"title":"Computational analysis of super-resolved in situ sequencing data reveals genes modified by immune-tumor contact events","authors":"Michal Danino-Levi, Tal Goldberg, Maya Keter, Nikol Akselrod, Noa Shprach-Buaron, Modi Safra, Gonen Singer, Shahar Alon","doi":"10.1261/rna.079801.123","DOIUrl":"https://doi.org/10.1261/rna.079801.123","url":null,"abstract":"Cancer cells can manipulate immune cells and escape from the immune system response. Quantifying the molecular changes that occur when an immune cell is touching a tumor cell can increase our understanding of the underlying mechanisms. Recently, it became possible to perform such measurements in situ, for example using expansion sequencing, which enabled in situ sequencing of genes with super-resolution. We systematically examined whether individual immune cells from specific cell types express genes differently when in physical proximity to individual tumor cells. First, we demonstrated that a dense mapping of genes in situ can be utilized for the segmentation of cell bodies in 3D, thus improving our ability to detect likely touching cells. Next, we utilized three different computational approaches to detect the molecular changes that are triggered by proximity: differential expression analysis, tree-based machine learning classifiers, and matrix factorization analysis. This systematic analysis revealed tens of genes, in specific cell types, whose expression separates immune cells that are proximal to tumor cells from those that are not proximal, with a significant overlap between the different detection methods. Remarkably, an order of magnitude more genes are trigger by proximity to tumor cells in CD8 T cells compared to CD4 T cells, in line with the ability of CD8 T cells to directly bind Major Histocompatibility Complex (MHC) Class I on tumor cells. Thus, in situ sequencing of an individual biopsy can be used to detect genes likely involved in immune-tumor cell-cell interactions. The data used in this manuscript and the code of the InSituSeg, Machine learning, cNMF and Moran’s I methods are publicly available at https://zenodo.org/record/7845775 (DOI: 10.5281/zenodo.7845775).","PeriodicalId":21401,"journal":{"name":"RNA","volume":"48 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140589619","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

Contribution of an alternative 16S rRNA helix to biogenesis of the 30S subunit of the ribosome 另一种 16S rRNA 螺旋对核糖体 30S 亚基生物形成的贡献

IF 4.5 3区生物学

RNA Pub Date : 2024-04-03 DOI: 10.1261/rna.079960.124

Benjamin R. Warner, Kurt Fredrick

{"title":"Contribution of an alternative 16S rRNA helix to biogenesis of the 30S subunit of the ribosome","authors":"Benjamin R. Warner, Kurt Fredrick","doi":"10.1261/rna.079960.124","DOIUrl":"https://doi.org/10.1261/rna.079960.124","url":null,"abstract":"30S subunits become inactive upon exposure to low Mg2+ concentration, due to a reversible conformational change that entails nucleotides (nt) in the neck helix (h28) and 3’ tail of 16S rRNA. This active-to-inactive transition involves partial unwinding of h28 and re-pairing of nt 921-923 with nt 1532-1534, which requires flipping of the 3’ tail by ~180 degrees. Growing evidence suggests that immature 30S particles adopt the inactive conformation in the cell, and transition to the active state occurs at a late stage of maturation. Here, we target nucleotides that form the alternative helix (hALT) of the inactive state. Using an orthogonal ribosome system, we find that disruption of hALT decreases translation activity in the cell modestly, by ~2-fold, without compromising ribosome fidelity. Ribosomes carrying substitutions at positions 1532-1533 support growth of E. coli strain Δ7 prrn (which carries a single rRNA operon), albeit at rates 10-20% slower than wild-type ribosomes. These mutant Δ7 prrn strains accumulate free 30S particles and precursor 17S rRNA, indicative of biogenesis defects. Analysis of purified control and mutant subunits suggests that hALT stabilizes the inactive state by 1.2 kcal/mol with little-to-no impact on the active state or the transition state of conversion.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"28 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140589170","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

Robo-Therm, a pipeline to RNA Thermometer discovery and validation Robo-Therm，发现和验证 RNA 温度计的管道

IF 4.5 3区生物学

RNA Pub Date : 2024-04-02 DOI: 10.1261/rna.079980.124

Davis M Sharts, Maria T. Almanza, Andrea Victoria Banks, Alyssa M Castellanos, Catherine G O. Hernandez, Monica L Lopez, Daniela Rodriguez, Alina Y Tong, Maximilian R Segeberg, Luiz F. M. Passalacqua, Michael M Abdelsayed

{"title":"Robo-Therm, a pipeline to RNA Thermometer discovery and validation","authors":"Davis M Sharts, Maria T. Almanza, Andrea Victoria Banks, Alyssa M Castellanos, Catherine G O. Hernandez, Monica L Lopez, Daniela Rodriguez, Alina Y Tong, Maximilian R Segeberg, Luiz F. M. Passalacqua, Michael M Abdelsayed","doi":"10.1261/rna.079980.124","DOIUrl":"https://doi.org/10.1261/rna.079980.124","url":null,"abstract":"RNA thermometers are highly structured noncoding RNAs located in the 5′ untranslated regions (UTR) of genes that regulate expression by undergoing conformational changes in response to temperature. The discovery of RNA thermometers through bioinformatics is difficult because there is little sequence conservation among their structural elements. Thus, the abundance of these thermo-sensitive regulatory structures remains unclear. Herein, to advance the discovery and validation of RNA thermometers, we developed Robo-Therm, a pipeline that combines an adaptive and user-friendly in silico motif search with a well-established reporter system. Through our application of Robo-Therm, we discovered two novel RNA thermometers in bacterial and bacteriophage genomes found in the human gut. One of these thermometers is present in 5′-UTR of a gene that codes for σ70 RNA polymerase subunit in the bacteria Mediterraneibacter gnavus and Bacteroides pectinophilus, and in the bacteriophage Caudoviricetes, which infects Bacteroides pectinophilus. The other thermometer is in the 5′-UTR of a tetracycline resistance gene (tetR) in the intestinal bacteria Escherichia coli and Shigella flexneri. Our Robo-Therm pipeline can be applied to discover multiple RNA thermometers across various genomes.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"121 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140603407","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

NMR characterization and ligand binding site of the stem loop 2 motif (s2m) from the Delta variant of SARS-CoV-2 SARS-CoV-2 三角洲变体茎环 2 节点 (s2m) 的核磁共振特征和配体结合位点

IF 4.5 3区生物学

RNA Pub Date : 2024-04-02 DOI: 10.1261/rna.079902.123

Tobias Matzel, Maria Wirtz Martin, Alexander Herr, Anna Wacker, Christian Richter, Sridhar Sreeramulu, Harald Schwalbe

{"title":"NMR characterization and ligand binding site of the stem loop 2 motif (s2m) from the Delta variant of SARS-CoV-2","authors":"Tobias Matzel, Maria Wirtz Martin, Alexander Herr, Anna Wacker, Christian Richter, Sridhar Sreeramulu, Harald Schwalbe","doi":"10.1261/rna.079902.123","DOIUrl":"https://doi.org/10.1261/rna.079902.123","url":null,"abstract":"The stem loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3’-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756 mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR analysis. With Delta, a variant of concern has evolved with one mutation compared to the original sequence that replaces G29742 by U29742. We show here that this mutation results in a more defined structure at ambient temperature accompanied by a rise in melting temperature. Consequently, we were able to identify over 90 % of the relevant NMR resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as 4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure, (sub-) nanosecond dynamics and ribose conformation of s2m Delta based on heteronuclear 13C NOE and T1 measurements and ribose carbon chemical shift-derived canonical coordinates. We further show that the G29742U mutation in Delta has no influence on the druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we identify the flexible regions of s2m as primary site for small molecule binding.","PeriodicalId":21401,"journal":{"name":"RNA","volume":"23 1","pages":""},"PeriodicalIF":4.5,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140589169","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: Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells 更正：SRPK1对SRSF1的磷酸化调节结直肠细胞中肿瘤相关Rac1b的替代剪接

IF 4.5 3区生物学

RNA Pub Date : 2024-04-01 DOI: 10.1261/rna.079955.123

Vânia Gonçalves, Andreia Henriques, Joana Pereira, Ana Neves Costa, Mary Pat Moyer, Luís Ferreira Moita, Margarida Gama-Carvalho, Paulo Matos, Peter Jordan

引用次数: 0

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