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The Journal of Biological Chemistry Jan 2024Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases...
Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.
Topics: Cyclins; Molecular Conformation; Polyadenylation; Humans; Cyclin-Dependent Kinases
PubMed: 38016516
DOI: 10.1016/j.jbc.2023.105501 -
International Journal of Molecular... Mar 2023Precursor message RNA requires processing to generate mature RNA. Cleavage and polyadenylation at the 3'-end in the maturation of mRNA is one of key processing steps in... (Review)
Review
Precursor message RNA requires processing to generate mature RNA. Cleavage and polyadenylation at the 3'-end in the maturation of mRNA is one of key processing steps in eukaryotes. The polyadenylation (poly(A)) tail of mRNA is an essential feature that is required to mediate its nuclear export, stability, translation efficiency, and subcellular localization. Most genes have at least two mRNA isoforms via alternative splicing (AS) or alternative polyadenylation (APA), which increases the diversity of transcriptome and proteome. However, most previous studies have focused on the role of alternative splicing on the regulation of gene expression. In this review, we summarize the recent advances concerning APA in the regulation of gene expression and in response to stresses in plants. We also discuss the mechanisms for the regulation of APA for plants in the adaptation to stress responses, and suggest that APA is a novel strategy for the adaptation to environmental changes and response to stresses in plants.
Topics: Polyadenylation; Alternative Splicing; RNA; Transcriptome; RNA, Messenger; Plants; Gene Expression Regulation; 3' Untranslated Regions
PubMed: 36902157
DOI: 10.3390/ijms24054727 -
Cell Reports Mar 2024The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two,...
The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two, WDR33v2 (V2) and WDR33v3 (V3), have not been studied. Here, we report that V2 and V3 are generated by alternative polyadenylation, and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly, V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING), the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-β induction by preventing STING disulfide oligomerization but promotes autophagy, likely by recruiting WIPI2 isoforms. V3, on the other hand, functions to increase STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated, indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated.
Topics: Humans; mRNA Cleavage and Polyadenylation Factors; RNA, Messenger; Polyadenylation; Membrane Proteins; Protein Isoforms; Immunity, Innate
PubMed: 38430516
DOI: 10.1016/j.celrep.2024.113886 -
Nature Communications Feb 2023Alternative polyadenylation (APA) plays an essential role in brain development; however, current transcriptome-wide association studies (TWAS) largely overlook APA in...
Alternative polyadenylation (APA) plays an essential role in brain development; however, current transcriptome-wide association studies (TWAS) largely overlook APA in nominating susceptibility genes. Here, we performed a 3' untranslated region (3'UTR) APA TWAS (3'aTWAS) for 11 brain disorders by combining their genome-wide association studies data with 17,300 RNA-seq samples across 2,937 individuals. We identified 354 3'aTWAS-significant genes, including known APA-linked risk genes, such as SNCA in Parkinson's disease. Among these 354 genes, ~57% are not significant in traditional expression- and splicing-TWAS studies, since APA may regulate the translation, localization and protein-protein interaction of the target genes independent of mRNA level expression or splicing. Furthermore, we discovered ATXN3 as a 3'aTWAS-significant gene for amyotrophic lateral sclerosis, and its modulation substantially impacted pathological hallmarks of amyotrophic lateral sclerosis in vitro. Together, 3'aTWAS is a powerful strategy to nominate important APA-linked brain disorder susceptibility genes, most of which are largely overlooked by conventional expression and splicing analyses.
Topics: Humans; Polyadenylation; Transcriptome; Amyotrophic Lateral Sclerosis; Genome-Wide Association Study; Parkinson Disease; 3' Untranslated Regions
PubMed: 36737438
DOI: 10.1038/s41467-023-36311-8 -
Proceedings of the National Academy of... Dec 2022Hexanucleotide GC repeat expansions in the gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat...
Hexanucleotide GC repeat expansions in the gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing GC repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged mice. However, chronic administration of anti-GA antibodies in AAV-(GC) mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.
Topics: Animals; Humans; Mice; Antigen-Antibody Complex; C9orf72 Protein; Dipeptides; Disease Models, Animal; Genes, Regulator; Poly A
PubMed: 36454749
DOI: 10.1073/pnas.2123487119 -
Wiley Interdisciplinary Reviews. RNA Mar 2021Termini often determine the fate of RNA molecules. In recent years, 3' ends of almost all classes of RNA species have been shown to acquire nontemplated nucleotides that... (Review)
Review
Termini often determine the fate of RNA molecules. In recent years, 3' ends of almost all classes of RNA species have been shown to acquire nontemplated nucleotides that are added by terminal nucleotidyltransferases (TENTs). The best-described role of 3' tailing is the bulk polyadenylation of messenger RNAs in the cell nucleus that is catalyzed by canonical poly(A) polymerases (PAPs). However, many other enzymes that add adenosines, uridines, or even more complex combinations of nucleotides have recently been described. This review focuses on metazoan TENTs, which are either noncanonical PAPs or terminal uridylyltransferases with varying processivity. These enzymes regulate RNA stability and RNA functions and are crucial in early development, gamete production, and somatic tissues. TENTs regulate gene expression at the posttranscriptional level, participate in the maturation of many transcripts, and protect cells against viral invasion and the transposition of repetitive sequences. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.
Topics: Animals; Nucleotidyltransferases; Polyadenylation; RNA; RNA Stability; RNA, Messenger
PubMed: 33145994
DOI: 10.1002/wrna.1622 -
Bioscience Reports Mar 2023RNA is a fundamental biomolecule that has many purposes within cells. Due to its single-stranded and flexible nature, RNA naturally folds into complex and dynamic... (Review)
Review
RNA is a fundamental biomolecule that has many purposes within cells. Due to its single-stranded and flexible nature, RNA naturally folds into complex and dynamic structures. Recent technological and computational advances have produced an explosion of RNA structural data. Many RNA structures have regulatory and functional properties. Studying the structure of nascent RNAs is particularly challenging due to their low abundance and long length, but their structures are important because they can influence RNA processing. Precursor RNA processing is a nexus of pathways that determines mature isoform composition and that controls gene expression. In this review, we examine what is known about human nascent RNA structure and the influence of RNA structure on processing of precursor RNAs. These known structures provide examples of how other nascent RNAs may be structured and show how novel RNA structures may influence RNA processing including splicing and polyadenylation. RNA structures can be targeted therapeutically to treat disease.
Topics: Humans; RNA Precursors; RNA Splicing; RNA; Polyadenylation; Gene Expression
PubMed: 36689327
DOI: 10.1042/BSR20220149 -
Nature Communications Jun 2023Minimally invasive biodegradable implants with regeneration have been a frontier trend in clinic. Degeneration of nucleus pulposus (NP) is irreversible in most of spine...
Minimally invasive biodegradable implants with regeneration have been a frontier trend in clinic. Degeneration of nucleus pulposus (NP) is irreversible in most of spine diseases, and traditional spinal fusion or discectomy usually injure adjacent segments. Here, an innovative minimally invasive biodegradable NP scaffold with function regeneration inspired by cucumber tendril is developed using shape memory polymer poly(glycerol-dodecanoate) (PGD), whose mechanical property is controlled to the similar with human NP by adjusting synthetic parameters. The chemokine stromal cell-derived factor-1α (SDF-1α) is immobilized to the scaffold recruiting autologous stem cells from peripheral tissue, which has better ability of maintaining disc height, recruiting autologous stem cells, and inducing regeneration of NP in vivo compared to PGD without chemokine group and hydrogel groups significantly. It provides an innovative way to design minimally invasive implants with biodegradation and functional recovery, especially for irreversible tissue injury, including NP, cartilage and so on.
Topics: Humans; Glycerol; Nucleus Pulposus; Absorbable Implants; Biodegradation, Environmental; Poly A
PubMed: 37391454
DOI: 10.1038/s41467-023-39604-0 -
Immunological Reviews Nov 2021The latest advances in next-generation sequencing studies and transcriptomic profiling over the past decade have highlighted a surprising frequency of genes regulated by... (Review)
Review
The latest advances in next-generation sequencing studies and transcriptomic profiling over the past decade have highlighted a surprising frequency of genes regulated by RNA processing mechanisms in the immune system. In particular, two control steps in mRNA maturation, namely alternative splicing and alternative polyadenylation, are now recognized to occur in the vast majority of human genes. Both have the potential to alter the identity of the encoded protein, as well as control protein abundance or even protein localization or association with other factors. In this review, we will provide a summary of the general mechanisms by which alternative splicing (AS) and alternative polyadenylation (APA) occur, their regulation within cells of the immune system, and their impact on immunobiology. In particular, we will focus on how control of apoptosis by AS and APA is used to tune cell fate during an immune response.
Topics: 3' Untranslated Regions; Alternative Splicing; Apoptosis; Humans; Immunity; Polyadenylation; RNA, Messenger
PubMed: 34368964
DOI: 10.1111/imr.13018 -
Nucleic Acids Research Jan 2021Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism that recognizes different polyadenylation signals on transcripts, resulting...
Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism that recognizes different polyadenylation signals on transcripts, resulting in transcripts with different lengths of 3' untranslated regions and thereby influencing a series of biological processes. Recent studies have highlighted the important roles of APA in human. However, APA profiles in other animals have not been fully recognized, and there is no database that provides comprehensive APA information for other animals except human. Here, by using the RNA sequencing data collected from public databases, we systematically characterized the APA profiles in 9244 samples of 18 species. In total, we identified 342 952 APA events with a median of 17 020 per species using the DaPars2 algorithm, and 315 691 APA events with a median of 17 953 per species using the QAPA algorithm in these 18 species, respectively. In addition, we predicted the polyadenylation sites (PAS) and motifs near PAS of these species. We further developed Animal-APAdb, a user-friendly database (http://gong_lab.hzau.edu.cn/Animal-APAdb/) for data searching, browsing and downloading. With comprehensive information of APA events in different tissues of different species, Animal-APAdb may greatly facilitate the exploration of animal APA patterns and novel mechanisms, gene expression regulation and APA evolution across tissues and species.
Topics: 3' Untranslated Regions; Alternative Splicing; Animals; Computational Biology; Databases, Genetic; Humans; Nucleotide Motifs; Polyadenylation; RNA, Messenger; Software; Web Browser
PubMed: 32986825
DOI: 10.1093/nar/gkaa778