scholarly journals The RNA demethylase FTO targets m6Am in snRNA to establish distinct methyl isoforms that influence splicing

2018 ◽  
Author(s):  
Jan Mauer ◽  
Miriam Sindelar ◽  
Théo Guez ◽  
Jean-Jacques Vasseur ◽  
Andrea Rentmeister ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Mrna Splicing ◽  
Rna Modifications ◽  
Methylation State ◽  
Nucleotide Modification ◽  
Small Nuclear Rnas ◽  
Potential Link ◽  
Endogenous Metabolites

SummarySmall nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. During their biogenesis, snRNAs acquire several constitutive nucleotide modifications. Here we show that snRNAs also contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1-isoform with a single-methylated adenosine (2’-O-methyladenosine, Am), which is then converted to a dimethylated m2-isoform (N6,2’-O-dimethyladenosine, m6Am). The relative m1- and m2-isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2-isoform. We show FTO is inhibited by endogenous metabolites, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO has a central role in snRNA biogenesis and controls a previously unknown step of snRNA processing involving reversible methylation, thereby providing a potential link between reversible RNA modifications and mRNA splicing.

scholarly journals Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

2021 ◽  
Author(s):  
Jonathan Bizarro ◽  
Svetlana Deryusheva ◽  
Ludivine Wacheul ◽  
Varun Gupta ◽  
Felix G.M. Ernst ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Small Molecule ◽  
Mode Of Action ◽  
Hematological Malignancies ◽  
Mrna Splicing ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Small Nuclear Rnas ◽  
Splicing Patterns ◽  
Ck2 Inhibitors

ABSTRACTSpliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2’-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

scholarly journals Spliceosomal snRNA Epitranscriptomics

2021 ◽  
Vol 12 ◽  
Author(s):  
Pedro Morais ◽  
Hironori Adachi ◽  
Yi-Tao Yu
Keyword(s):  
Cell Nucleus ◽  
Current Knowledge ◽  
Protein Complexes ◽  
Mrna Splicing ◽  
Rna Modifications ◽  
Small Nuclear Rnas ◽  
Sequence Elements ◽  
Critical Components ◽  
Rna Protein Complexes ◽  
Small Nuclear Ribonucleoproteins

Small nuclear RNAs (snRNAs) are critical components of the spliceosome that catalyze the splicing of pre-mRNA. snRNAs are each complexed with many proteins to form RNA-protein complexes, termed as small nuclear ribonucleoproteins (snRNPs), in the cell nucleus. snRNPs participate in pre-mRNA splicing by recognizing the critical sequence elements present in the introns, thereby forming active spliceosomes. The recognition is achieved primarily by base-pairing interactions (or nucleotide-nucleotide contact) between snRNAs and pre-mRNA. Notably, snRNAs are extensively modified with different RNA modifications, which confer unique properties to the RNAs. Here, we review the current knowledge of the mechanisms and functions of snRNA modifications and their biological relevance in the splicing process.

scholarly journals Genome-wide Analysis of Alternative Pre-mRNA Splicing

2007 ◽  
Vol 283 (3) ◽  
pp. 1229-1233 ◽  
Author(s):  
Claudia Ben-Dov ◽  
Britta Hartmann ◽  
Josefin Lundgren ◽  
Juan Valcárcel
Keyword(s):  
Alternative Splicing ◽  
Large Scale ◽  
Mrna Splicing ◽  
Diagnostic Tools ◽  
Primary Transcript ◽  
Genome Wide ◽  
Human Genes ◽  
Multicellular Organisms ◽  
Eukaryotic Genomes ◽  
Key Questions

Alternative splicing of mRNA precursors allows the synthesis of multiple mRNAs from a single primary transcript, significantly expanding the information content and regulatory possibilities of higher eukaryotic genomes. High-throughput enabling technologies, particularly large-scale sequencing and splicing-sensitive microarrays, are providing unprecedented opportunities to address key questions in this field. The picture emerging from these pioneering studies is that alternative splicing affects most human genes and a significant fraction of the genes in other multicellular organisms, with the potential to greatly influence the evolution of complex genomes. A combinatorial code of regulatory signals and factors can deploy physiologically coherent programs of alternative splicing that are distinct from those regulated at other steps of gene expression. Pre-mRNA splicing and its regulation play important roles in human pathologies, and genome-wide analyses in this area are paving the way for improved diagnostic tools and for the identification of novel and more specific pharmaceutical targets.

scholarly journals Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan

1993 ◽  
Vol 291 (2) ◽  
pp. 361-367 ◽  
Author(s):  
J Grover ◽  
P J Roughley
Keyword(s):  
Alternative Splicing ◽  
Articular Cartilage ◽  
Regulatory Protein ◽  
Mrna Splicing ◽  
Human Articular Cartilage ◽  
Complement Regulatory Protein ◽  
Mature Adult ◽  
Epidermal Growth ◽  
Egf Domain

The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture.

Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract

1991 ◽  
Vol 11 (7) ◽  
pp. 3425-3431
Author(s):  
P Delannoy ◽  
M H Caruthers
Keyword(s):  
Heat Treatment ◽  
Hela Cell ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Nuclear Extracts ◽  
Small Nuclear Rnas ◽  
A Minor ◽  
Cell Nuclear Extract

Mild heat treatment of HeLa cell nuclear extracts (NE) selectively inhibits pre-mRNA splicing. Heat-inactivated extracts can be complemented by a small amount of untreated NE. Utilizing this complementation assay and a combination of ion-exchange, affinity, and hydrophobic chromatography, a heat reversal factor (HRF) was purified from NE that is required to rescue pre-mRNA splicing from a heat-inactivated extract. This activity in its most purified form consistently copurified in a fraction containing two 70-kDa proteins and a minor polypeptide of approximately 100 kDa. It was free of the major small nuclear RNAs, sensitive to protease, and required to rescue spliceosome formation from a heat-inactivated nuclear extract. These results suggest that this factor is a protein that may be an important component in pre-mRNA splicing, or alternatively, it may be involved in renaturation of a heat-sensitive splicing factor.

scholarly journals U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

1993 ◽  
Vol 13 (5) ◽  
pp. 2666-2676 ◽  
Author(s):  
J B Cohen ◽  
S D Broz ◽  
A D Levinson
Keyword(s):  
Splice Site ◽  
Mammalian Cells ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
Gene Complementation ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577 ◽  
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

scholarly journals Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

2017 ◽  
Author(s):  
Adriana Buskin ◽  
Lili Zhu ◽  
Valeria Chichagova ◽  
Basudha Basu ◽  
Sina Mozaffari-Jovin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Retinitis Pigmentosa ◽  
Gene Editing ◽  
Autosomal Dominant ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Patient Specific ◽  
Rpe Cells ◽  
Autosomal Dominant Retinitis Pigmentosa

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

scholarly journals hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

2021 ◽  
Author(s):  
Shuiqiao Yuan ◽  
Shenglei Feng ◽  
Jinmei Li ◽  
Hui Wen ◽  
Kuan Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Cell Development ◽  
Mrna Splicing ◽  
Conditional Knockout ◽  
Cell Junction ◽  
Germ Cell Development ◽  
Splicing Regulators

Abstract Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

Sign in / Sign up

Export Citation Format

Share Document