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Biochemical and Biophysical Research... Jun 2024Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3' end of human tRNA. The primer tRNA is selectively packaged into the virus in...
Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3' end of human tRNA. The primer tRNA is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5' leader (5'L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNA for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5'L responsible for LysRS binding, how the conformations and various hairpin structures of 5'L regulate 5'L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5'L significantly influences its binding affinity with LysRS. The 5'L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5'L conformation that is not selected for packaging. Additionally, dimerization of 5'L impairs LysRS-5'L interaction. Furthermore, among various regions of 5'L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5'L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.
PubMed: 38878758
DOI: 10.1016/j.bbrc.2024.150252 -
Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.BMC Plant Biology Jun 2024The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to...
BACKGROUND
The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce.
RESULTS
Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction.
CONCLUSION
We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.
Topics: Arabidopsis Proteins; Arabidopsis; RNA-Binding Proteins; Proteome; RNA, Messenger; RNA, Plant; Gene Expression Regulation, Plant
PubMed: 38877390
DOI: 10.1186/s12870-024-05249-4 -
Nucleic Acids Research Jun 2024The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail...
The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.
PubMed: 38874498
DOI: 10.1093/nar/gkae510 -
Experimental & Molecular Medicine Jun 2024Circular RNAs (circRNAs) are covalently closed single-stranded RNAs without a 5' cap structure and a 3' poly(A) tail typically present in linear mRNAs of eukaryotic... (Review)
Review
Circular RNAs (circRNAs) are covalently closed single-stranded RNAs without a 5' cap structure and a 3' poly(A) tail typically present in linear mRNAs of eukaryotic cells. CircRNAs are predominantly generated through a back-splicing process within the nucleus. CircRNAs have long been considered non-coding RNAs seemingly devoid of protein-coding potential. However, many recent studies have challenged this idea and have provided substantial evidence that a subset of circRNAs can associate with polysomes and indeed be translated. Therefore, in this review, we primarily highlight the 5' cap-independent internal initiation of translation that occurs on circular RNAs. Several molecular features of circRNAs, including the internal ribosome entry site, N-methyladenosine modification, and the exon junction complex deposited around the back-splicing junction after back-splicing event, play pivotal roles in their efficient internal translation. We also propose a possible relationship between the translatability of circRNAs and their stability, with a focus on nonsense-mediated mRNA decay and nonstop decay, both of which are well-characterized mRNA surveillance mechanisms. An in-depth understanding of circRNA translation will reshape and expand our current knowledge of proteomics.
PubMed: 38871818
DOI: 10.1038/s12276-024-01220-3 -
Methods in Molecular Biology (Clifton,... 2024Recent advancements in detection and mapping methods have enabled researchers to uncover the biological importance of RNA chemical modifications, which play a vital role...
Recent advancements in detection and mapping methods have enabled researchers to uncover the biological importance of RNA chemical modifications, which play a vital role in post-transcriptional gene regulation. Although numerous types of RNA modifications have been identified in higher eukaryotes, only a few have been extensively studied for their biological functions. Of these, N-methyladenosine (mA) is the most prevalent and important mRNA modification that influences various aspects of RNA metabolism, including mRNA stability, degradation, splicing, alternative polyadenylation, export, and localization, as well as translation. Thus, they have implications for a variety of biological processes, including growth, development, and stress responses. The mA deposition or removal on transcripts is dynamic and is altered in response to internal and external cues. Because this mark can alter gene expression under stress conditions, it is essential to identify the transcripts that can acquire or lose this epitranscriptomic mark upon exposure to stress conditions. Here we describe a step-by-step protocol for identifying stress-responsive transcriptome-wide mA changes using RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq).
Topics: Adenosine; Stress, Physiological; Transcriptome; Gene Expression Regulation, Plant; RNA, Plant; High-Throughput Nucleotide Sequencing; RNA, Messenger; Gene Expression Profiling; Arabidopsis; Sequence Analysis, RNA; Immunoprecipitation; Plants; RNA Processing, Post-Transcriptional
PubMed: 38869786
DOI: 10.1007/978-1-0716-3973-3_3 -
Concerted action of ataxin-2 and PABPC1-bound mRNA poly(A) tail in the formation of stress granules.Nucleic Acids Research Jun 2024Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules...
Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules (SGs). While the mechanism behind stress-induced global PAT stabilization has recently emerged, the biological significance of PAT stabilization under stress remains elusive. Here, we demonstrate that stress-induced PAT stabilization is a prerequisite for SG formation. Perturbations in PAT length impact SG formation; PAT shortening, achieved by overexpressing mRNA deadenylases, inhibits SG formation, whereas PAT lengthening, achieved by overexpressing their dominant negative mutants or downregulating deadenylases, promotes it. PABPC1, which specifically binds to the PAT, is crucial for SG formation. Complementation analyses reveal that the PABC/MLLE domain of PABPC1, responsible for binding PAM2 motif-containing proteins, plays a key role. Among them, ataxin-2 is a known SG component. A dominant-negative approach reveals that the PAM2 motif of ataxin-2 is essential for SG formation. Notably, ataxin-2 increases stress sensitivity, lowering the threshold for SG formation, probably by promoting the aggregation of PABPC1-bound mRNA. The C-terminal region is responsible for the self-aggregation of ataxin-2. These findings underscore the critical roles of mRNA PAT, PABPC1 and ataxin-2 in SG formation and provide mechanistic insights into this process.
PubMed: 38869059
DOI: 10.1093/nar/gkae497 -
Autoimmunity Dec 2024Rheumatoid arthritis (RA) is the predominant manifestation of inflammatory arthritis, distinguished by an increasing burden of morbidity and mortality. The intricate...
Rheumatoid arthritis (RA) is the predominant manifestation of inflammatory arthritis, distinguished by an increasing burden of morbidity and mortality. The intricate interplay of genes and signalling pathways involved in synovial inflammation in patients with RA remains inadequately comprehended. This study aimed to ascertain the role of necroptosis in RA, as along with their associations with immune cell infiltration. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were employed to identify central genes for RA. In this study, identified total of 28 differentially expressed genes (DEGs) were identified in RA. Utilising WGCNA, two co-expression modules were generated, with one module demonstrating the strongest correlation with RA. Through the integration of differential gene expression analysis, a total of 5 intersecting genes were discovered. These 5 hub genes, namely fused in sarcoma (FUS), transformer 2 beta homolog (TRA2B), eukaryotic translation elongation factor 2 (EEF2), cleavage and polyadenylation specific factor 6 (CPSF6) and signal transducer and activator of transcription 3 (STAT3) were found to possess significant diagnostic value as determined by receiver operating characteristic (ROC) curve analysis. The close association between the concentrations of various immune cells is anticipated to contribute to the diagnosis and treatment of RA. Furthermore, the infiltration of immune cells mentioned earlier is likely to exert a substantial influence on the initiation of this disease.
Topics: Arthritis, Rheumatoid; Humans; Necroptosis; Gene Regulatory Networks; Gene Expression Profiling; Transcriptome; Computational Biology; Gene Expression Regulation; Signal Transduction; STAT3 Transcription Factor; Biomarkers; ROC Curve
PubMed: 38869013
DOI: 10.1080/08916934.2024.2358069 -
Molecular Cell Jun 2024The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear....
The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.
Topics: Arabidopsis; Arabidopsis Proteins; Gene Silencing; Gene Expression Regulation, Plant; Histones; RNA Polymerase II; Polycomb Repressive Complex 2; MADS Domain Proteins; Transcription, Genetic; Polyadenylation; Histone Demethylases; Transcription Termination, Genetic; Chromatin; RNA-Binding Proteins
PubMed: 38851186
DOI: 10.1016/j.molcel.2024.05.014 -
Molecular Cell Jun 2024The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the...
The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.
Topics: Arabidopsis Proteins; Arabidopsis; Chromatin; Gene Expression Regulation, Plant; MADS Domain Proteins; Transcription Termination, Genetic; RNA Polymerase II; Gene Silencing; Phosphoprotein Phosphatases; mRNA Cleavage and Polyadenylation Factors; Histones; Histone Deacetylases
PubMed: 38851185
DOI: 10.1016/j.molcel.2024.05.016 -
Nucleic Acids Research Jun 2024Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu...
Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing mRNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINEs use strategies to 'hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.
PubMed: 38850156
DOI: 10.1093/nar/gkae448