Role of TFIP11 in U6 snRNA modification and splicing efficiency
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA657749 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12465906_1.fastq.gz | Fastqsanger.gz | |||
| SRR12465906_2.fastq.gz | Fastqsanger.gz | |||
| SRR12465907_1.fastq.gz | Fastqsanger.gz | |||
| SRR12465907_2.fastq.gz | Fastqsanger.gz | |||
| SRR12465908_1.fastq.gz | Fastqsanger.gz |
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Project description:THUMPD2 installs N2-methylguanosine (m2G) at position 72 of the U6 snRNA, and this modification, which lies at the catalytic core of the spliceosome, is required for efficient splicing of weak splice sites. However, when during the U6 lifecycle this modification is installed and what features of the U6 snRNA and THUMPD2 are required for methylation of G72 remain elusive. Here, we show that U6 associated with THUMPD2 is not oligouridylated and that late-acting U6 snRNP biogenesis factors are enriched with THUMPD2. We map RNA crosslinking sites within the N-terminal region and C-terminal Rossman-fold methyltransferase domain of THUMPD2, and show the requirement of these domains for THUMPD2 interaction with U6 in cells. Using a newly developed m2G-sensitive deoxyribozyme to monitor U6-m2G72 levels in cellular RNAs, we demonstrate the requirement of SAM binding and interaction with the THUMPD2 cofactor TRMT112 for efficient methylation. Our data further reveal that 2’-O-methylation of C62, C63 and A70 do not increase the affinity of THUMPD2 for the U6 internal stem-loop (ISL), but enhance m2G methylation of G72. Nucleotide substitutions within the loop region of the ISL that extend the stem similarly increase m2G72 installation, demonstrating that the stability of the ISL is key for methylation by THUMPD2. Together these results provide a new layer of understanding of how an RNA modification that fine-tunes pre-mRNA splicing is introduced.
2026-05-25 | PXD053510 | Pride
Project description:MMARY THUMPD2 installs N2-methylguanosine (m2G) at position 72 of the U6 snRNA, and this modification, which lies at the catalytic core of the spliceosome, is required for efficient splicing of weak splice sites. However, when during the U6 lifecycle this modification is installed and what features of the U6 snRNA and THUMPD2 are required for methylation of G72 remain elusive. Here, we show that U6 associated with THUMPD2 is not oligouridylated and that late-acting U6 snRNP biogenesis factors are enriched with THUMPD2. We map RNA crosslinking sites within the N-terminal region and C-terminal Rossman-fold methyltransferase domain of THUMPD2, and show the requirement of these domains for THUMPD2 interaction with U6 in cells. Using a newly developed m2G-sensitive deoxyribozyme to monitor U6-m2G72 levels in cellular RNAs, we demonstrate the requirement of SAM binding and interaction with the THUMPD2 cofactor TRMT112 for efficient methylation. Our data further reveal that 2’-O-methylation of C62, C63 and A70 do not increase the affinity of THUMPD2 for the U6 internal stem-loop (ISL), but enhance m2G methylation of G72. Nucleotide substitutions within the loop region of the ISL that extend the stem similarly increase m2G72 installation, demonstrating that the stability of the ISL is key for methylation by THUMPD2. Together these results provide a new layer of understanding of how an RNA modification that fine-tunes pre-mRNA splicing is introduced.
2026-05-25 | PXD053619 | Pride
Project description:How the relatively evolutionarily conserved spliceosome is able to manage the enormously expanded number of splicing events that occur in humans (~200,000 vs. ~400 reported for yeast) is not well understood. Here, we show deposition of one RNA modification-N2-methylguanosine (m2G)-on the G72 nucleoside of U6 snRNA (known to function as the catalytic center of the spliceosome) results in profoundly increased pre-mRNA splicing activity in human cells. This U6 m2G72 modification is conserved among vertebrates. Further, we demonstrate that THUMPD2 is the methyltransferase responsible for U6 m2G72 and show that it interacts with an auxiliary protein (TRMT112) to specifically recognize both sequence and structural elements of U6. THUMPD2 KO blocks U6 m2G72 and down-regulates the pre-mRNA splicing activity of major spliceosome, yielding thousands of changed alternative splicing events of endogenous pre-mRNAs. Notably, the aberrantly spliced pre-mRNA population of the THUMPD2 KO cells elicits the nonsense-mediated mRNA decay (NMD) pathway and restricts cell proliferation. We also show that THUMPD2-mediated control of the U6 m2G72 modification is associated with age-related macular degeneration and retinal function. Our study thus demonstrates how an RNA epigenetic modification of the major spliceosome differentially regulates global mRNA splicing and impacts physiology and disease.
2024-01-02 | GSE236689 | GEO
Project description:The post-transcriptional maturation of U6 snRNA 3′-end is important for spliceosomal assembly and RNA splicing, and is catalyzed by sequential enzymatic activities of TUT1 and USB1, that is, an oligo(U) tail is first added by TUT1 to the freshly transcribed U6 snRNA, and then trimmed by USB1 to generate mature U6 snRNA. However, biallelic inactivation of TUT1 or USB1 is linked to distinct human developmental disorders, and their physiological functions remain unknown. Here, using multiple genetically engineered mouse models, we show that Tut1, but unexpectedly not Usb1, is essential for the maintenance of the epiblast and neural stem cells in vivo. Loss of Tut1 weakens the interactions between essential splicing factors and U6 snRNA, causes defective RNA splicing, and triggers massive DNA damage and subsequent cell death. Importantly, cell death can be prevented by recombinant U6 snRNA containing an oligo(U) tail. These results challenge the current model of U6 snRNA 3′-end maturation and function. We propose that TUT1 is an essential U6 snRNA 3′-end maturation or repair factor, and discuss the implications of these results for understanding the etiologies of TUT1- and USB1-related human disorders.
2026-06-03 | GSE305663 | GEO
Project description:The post-transcriptional maturation of U6 snRNA 3′-end is important for spliceosomal assembly and RNA splicing, and is catalyzed by sequential enzymatic activities of TUT1 and USB1, that is, an oligo(U) tail is first added by TUT1 to the freshly transcribed U6 snRNA, and then trimmed by USB1 to generate mature U6 snRNA. However, biallelic inactivation of TUT1 or USB1 is linked to distinct human developmental disorders, and their physiological functions remain unknown. Here, using multiple genetically engineered mouse models, we show that Tut1, but unexpectedly not Usb1, is essential for the maintenance of the epiblast and neural stem cells in vivo. Loss of Tut1 weakens the interactions between essential splicing factors and U6 snRNA, causes defective RNA splicing, and triggers massive DNA damage and subsequent cell death. Importantly, cell death can be prevented by recombinant U6 snRNA containing an oligo(U) tail. These results challenge the current model of U6 snRNA 3′-end maturation and function. We propose that TUT1 is an essential U6 snRNA 3′-end maturation or repair factor, and discuss the implications of these results for understanding the etiologies of TUT1- and USB1-related human disorders.
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Project description:The 3’ end of most cellular U6 snRNA has a 2′,3′-cyclic phosphate group, a post-transcriptional modification thought to be essential for proper RNA splicing and cellular function. Recent studies have established that the biogenesis of 2′,3′-cyclic phosphate requires two enzymes: first TUT1 adds a poly(U) tail to the 3’ end of U6 snRNA, followed by USB1-catalyzed trimming of the poly(U) tail. Here, we use nine genetically engineered mouse models of Tut1 and Usb1 to show that, contrary to what was previously believed, Usb1 and thus 2′,3′-cyclic phosphate is dispensable for intra-embryonic cell lineages. We find that Tut1, but not Usb1, is essential for the maintenance of the epiblast and neural stem cells. Loss of Tut1 causes defective RNA splicing, triggering massive DNA damage and subsequent cell death. In contrast, loss of Usb1 primarily damages endothelial cells of the extra-embryonic vasculature in the yolk sac by both cell-intrinsic and -extrinsic mechanisms, leading to fetal growth restriction. These results demonstrate that TUT1-catalyzed U6 snRNA 3’ end oligouridylation, rather than 2′,3′-cyclic phosphate as previously thought, is required for proper RNA splicing. Further studies are warranted to elucidate how these different post-transcriptional modifications regulate the function of U6 snRNA in RNA splicing in development and disease.
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Project description:The 3’ end of most cellular U6 snRNA has a 2′,3′-cyclic phosphate group, a post-transcriptional modification thought to be essential for proper RNA splicing and cellular function. Recent studies have established that the biogenesis of 2′,3′-cyclic phosphate requires two enzymes: first TUT1 adds a poly(U) tail to the 3’ end of U6 snRNA, followed by USB1-catalyzed trimming of the poly(U) tail. Here, we use nine genetically engineered mouse models of Tut1 and Usb1 to show that, contrary to what was previously believed, Usb1 and thus 2′,3′-cyclic phosphate is dispensable for intra-embryonic cell lineages. We find that Tut1, but not Usb1, is essential for the maintenance of the epiblast and neural stem cells. Loss of Tut1 causes defective RNA splicing, triggering massive DNA damage and subsequent cell death. In contrast, loss of Usb1 primarily damages endothelial cells of the extra-embryonic vasculature in the yolk sac by both cell-intrinsic and -extrinsic mechanisms, leading to fetal growth restriction. These results demonstrate that TUT1-catalyzed U6 snRNA 3’ end oligouridylation, rather than 2′,3′-cyclic phosphate as previously thought, is required for proper RNA splicing. Further studies are warranted to elucidate how these different post-transcriptional modifications regulate the function of U6 snRNA in RNA splicing in development and disease.
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Project description:Mpn1 proteins are evolutionarily conserved exonucleases that modify spliceosomal U6 small nuclear RNAs (snRNAs) post-transcriptionally. Mutations in the human MPN1 gene are associated to the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Mpn1 deficiency leads to aberrant U6 3M-bM-^@M-^Y end processing and accelerated U6 decay through unknown molecular mechanisms. Here we show that in mpn1M-NM-^T fission yeast cells U6 is barely bound by the protective Lsm2-8 complex, undergoes extensive oligoadenylation and is degraded by the nuclear RNA exonuclease Rrp6 independently of the poly(A) polymerase Cid14/Trf4. Mpn1 processes U6 in a spliceosome-dependent manner, as mutant U6 molecules that fail to join the spliceosome are not substrates for Mpn1. Moreover, human U6atac, the U6-like snRNA of the minor spliceosome, is a novel substrate for hMpn1. We unveil mechanistic details of a new U6 degradation pathway and further corroborate the notion that inefficient canonical and minor pre-mRNA splicing promotes PN. the 3' termini of U6 or tagged-U6 species from the indicated mutant cells were compared to wt yeast strain
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