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Introduction: The U1 small nuclear RNA (snRNA) forms ribonucleoprotein particles (RNPs) such as U1 snRNP and U1-TAF15 snRNP. U1 snRNP is one of the most studied RNPs due to its critical role in pre-mRNA splicing in defining the 5' splice site (5'ss) of every exon through direct interactions with sequences at exon/intron junctions. Recent reports support the role of U1 snRNP in all steps of transcription, namely initiation, elongation, and termination. Functions of U1-TAF15 snRNP are less understood, though it associates with the transcription machinery and may modulate pre-mRNA splicing by interacting with the 5'ss and/or 5'ss-like sequences within the pre-mRNA. An anti-U1 antisense oligonucleotide (ASO) that sequesters the 5' end of U1 snRNA inhibits the functions of U1 snRNP, including transcription and splicing. However, it is not known if the inhibition of U1 snRNP influences post-transcriptional regulation of pre-mRNA splicing through deep intronic sequences. Methods: We examined the effect of an anti-U1 ASO that sequesters the 5' end of U1 snRNA on transcription and splicing of all internal exons of the spinal muscular atrophy (SMA) genes, SMN1 and SMN2. Our study was enabled by the employment of a multi-exon-skipping detection assay (MESDA) that discriminates against prematurely terminated transcripts. We employed an SMN2 super minigene to determine if anti-U1 ASO differently affects splicing in the context of truncated introns. Results: We observed substantial skipping of multiple internal exons of SMN1 and SMN2 triggered by anti-U1 treatment. Suggesting a role for U1 snRNP in interacting with deep intronic sequences, early exons of the SMN2 super minigene with truncated introns were resistant to anti-U1 induced skipping. Consistently, overexpression of engineered U1 snRNAs targeting the 5'ss of early SMN1 and SMN2 exons did not prevent exon skipping caused by anti-U1 treatment. Discussion: Our results uncover a unique role of the U1 snRNA-associated RNPs in splicing regulation executed through deep intronic sequences. Findings are significant for developing novel therapies for SMA based on deep intronic targets. [ABSTRACT FROM AUTHOR]

IntroductionThe U1 small nuclear RNA (snRNA) forms ribonucleoprotein particles (RNPs) such as U1 snRNP and U1-TAF15 snRNP. U1 snRNP is one of the most studied RNPs due to its critical role in pre-mRNA splicing in defining the 5′ splice site (5′ss) of every exon through direct interactions with sequences at exon/intron junctions. Recent reports support the role of U1 snRNP in all steps of transcription, namely initiation, elongation, and termination. Functions of U1-TAF15 snRNP are less understood, though it associates with the transcription machinery and may modulate pre-mRNA splicing by interacting with the 5′ss and/or 5′ss-like sequences within the pre-mRNA. An anti-U1 antisense oligonucleotide (ASO) that sequesters the 5′ end of U1 snRNA inhibits the functions of U1 snRNP, including transcription and splicing. However, it is not known if the inhibition of U1 snRNP influences post-transcriptional regulation of pre-mRNA splicing through deep intronic sequences.MethodsWe examined the effect of an anti-U1 ASO that sequesters the 5′ end of U1 snRNA on transcription and splicing of all internal exons of the spinal muscular atrophy (SMA) genes, SMN1 and SMN2. Our study was enabled by the employment of a multi-exon-skipping detection assay (MESDA) that discriminates against prematurely terminated transcripts. We employed an SMN2 super minigene to determine if anti-U1 ASO differently affects splicing in the context of truncated introns.ResultsWe observed substantial skipping of multiple internal exons of SMN1 and SMN2 triggered by anti-U1 treatment. Suggesting a role for U1 snRNP in interacting with deep intronic sequences, early exons of the SMN2 super minigene with truncated introns were resistant to anti-U1 induced skipping. Consistently, overexpression of engineered U1 snRNAs targeting the 5′ss of early SMN1 and SMN2 exons did not prevent exon skipping caused by anti-U1 treatment.DiscussionOur results uncover a unique role of the U1 snRNA-associated RNPs in splicing regulation executed through deep intronic sequences. Findings are significant for developing novel therapies for SMA based on deep intronic targets.

During spliceosome assembly, interactions that bring the 5' and 3' ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

Mutations in the CNOT1 gene lead to an incurable rare neurological disorder mainly manifested as a clinical spectrum of intellectual disability, developmental delay, seizures, and behavioral problems. In this study, we investigated a classical splice site variant of CNOT1 (c.1343+1G>T) associated with neurodevelopmental disorders, which was a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. To link CNOT1 dysfunction with the neurodevelopmental phenotype observed in a patient, in vitro minigene assay was used to verify the effect of CNOT1 gene splice site variant c.1343+1G>T on mRNA splicing. We also explored the impact of transient transfection introducing modified U1 snRNA on correcting the splicing variant. Through minigene expression in mammalian cells, we demonstrated that the variant induced complete exon 12 skipping, which explained the patient's clinical condition and provided additional genetic diagnosis evidence for the clinical significance of the variant. Moreover, we confirmed that the aberrant splice pattern could be partially corrected by the modified U1 snRNA at the mRNA level, which provided strong evidence for the therapeutic potential of modified U1 snRNA in neutralizing the hazardous effect of incorrect splicing patterns. [ABSTRACT FROM AUTHOR]

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases. [ABSTRACT FROM AUTHOR]

Autosomal dominant optic atrophy (ADOA) is frequently caused by mutations in the optic atrophy 1 (OPA1) gene, with haploinsufficiency being the major genetic pathomechanism. Almost 30% of the OPA1-associated cases suffer from splice defects. We identified a novel OPA1 mutation, c.1065+5G>A, in patients with ADOA. In patient-derived fibroblasts, the mutation led to skipping of OPA1 exon 10, reducing the OPA1 protein expression by approximately 50%. We developed a molecular treatment to correct the splice defect in OPA1 using engineered U1 splice factors retargeted to different locations in OPA1 exon 10 or intron 10. The strongest therapeutic effect was detected when U1 binding was engineered to bind to intron 10 at position +18, a position predicted by bioinformatics to be a promising binding site. We were able to significantly silence the effect of the mutation (skipping of exon 10) and simultaneously increase the expression level of normal transcripts. Retargeting U1 to the canonical splice donor site did not lead to a detectable splice correction. This proof-of-concept study indicates for the first time the feasibility of splice mutation correction as a treatment option for ADOA. Increasing the amount of correctly spliced OPA1 transcripts may suffice to overcome the haploinsufficiency.

• Engineering of sequence-specific alternative splicing modulators. • Robust exclusion of endogenous FAS exon 6 and inclusion of endogenous SMN2 exon 7. • Underscoring U1 snRNA as a candidate therapeutic and research tool. Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community. [ABSTRACT FROM AUTHOR]

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases.

RNA-binding proteins (RBPs) are involved in all aspects of RNA metabolism, and RNA-RBP interactions are important for cell homeostasis and viral replication. The global RNA-binding proteome was recently reported; however, little is known about the proteins that bind to specific RNAs. In this study, we describe a novel CRISPR-based RNA interaction proteomics method in live cells. In brief, dCas13a with an HA tag was expressed in cells and bound to an RNA of interest with the help of gRNA. The RNA-protein complexes physically bound to dCas13a-HA were crosslinked using UV light and captured using anti-HA beads, after which the proteins were purified and identified using mass spectrometry. We optimized this system and subsequently applied it to U1 small nuclear RNA, which revealed 226 proteins. • dCas13a based RNA binding protein detecting method in vivo. • The all-in-one plasmid simplifies the operation process. • CBRIP identified RBPs on U1 snRNA are highly specified. • RPL7 is verified to be an U1 snRNA binding protein. [ABSTRACT FROM AUTHOR]

The human U1 snRNA is encoded by a multigene family consisting of transcribed variants and defective pseudogenes. Many variant U1 (vU1) snRNAs have been demonstrated to not only be transcribed but also processed by the addition of a trimethylated guanosine cap, packaged into snRNPs, and assembled into spliceosomes; however, their capacity to facilitate pre-mRNA splicing has, so far, not been tested. A recent systematic analysis of the human snRNA genes identified 178 U1 snRNA genes that are present in the genome as either tandem arrays or single genes on multiple chromosomes. Of these, 15 were found to be expressed in human tissues and cell lines, although at significantly low levels from their endogenous loci, <0.001% of the canonical U1 snRNA. In this study, we found that placing the variants in the context of the regulatory elements of the RNU1-1 gene improves the expression of many variants to levels comparable to the canonical U1 snRNA. Application of a previously established HeLa cell-based minigene reporter assay to examine the capacity of the vU1 snRNAs to support pre-mRNA splicing revealed that even though the exogenously expressed variant snRNAs were enriched in the nucleus, only a few had a measurable effect on splicing., (© 2024 Wong et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS. [Display omitted] • ZC3H4, WDR82, and ARS2 form a complex and terminate common ncRNAs • WDR82 and ARS2 support ZC3H4 by different mechanisms • PNUTS universally controls transcriptional termination • U1 snRNA counteracts ZC3H4, WDR82, and PNUTS at protein-coding genes Estell et al. identify a restrictor complex of ZC3H4, WDR82, and ARS2 that acts together with PNUTS to terminate unproductive transcription. Coding transcripts are much less vulnerable to this "restriction" activity because their transcription elongation is more productive. This efficient elongation is supported by U1 snRNA. [ABSTRACT FROM AUTHOR]

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47 hi ) with impaired regenerative capacity that predominate with aging. The prevalent CD47 hi MuSC subset suppresses the residual functional CD47 lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging., Competing Interests: Declaration of interests H.M.B. is cofounder of Rejuvenation Technologies Inc. and Epirium Bio. H.M.B. and E.P. are named inventors on patent application no. PCT/US2021/038549 held by Stanford University., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Inherited retinal dystrophies are caused by mutations in more than 250 genes, each of them carrying several types of mutations that can lead to different clinical phenotypes. Mutations in Retinitis Pigmentosa GTPase-Regulator ( RPGR ) cause X-linked Retinitis pigmentosa (RP). A nucleotide substitution in intron 9 of RPGR causes the increase of an alternatively spliced isoform of the mature mRNA, bearing exon 9a (E9a). This introduces a stop codon, leading to truncation of the protein. Aiming at restoring impaired gene expression, we developed an antisense RNA-based therapeutic approach for the skipping of RPGR E9a. We designed a set of specific U1 antisense snRNAs (U1&#95;asRNAs) and tested their efficacy in vitro , upon transient cotransfection with RPGR minigene reporter systems in HEK-293T, 661W, and PC-12 cell lines. We thus identified three chimeric U1&#95;asRNAs that efficiently mediate E9a skipping, correcting the genetic defect. Unexpectedly, the U1-5'antisense construct, which exhibited the highest exon-skipping efficiency in PC-12 cells, induced E9a inclusion in HEK-293T and 661W cells, indicating caution in the choice of preclinical model systems when testing RNA splicing-correcting therapies. Our data provide a proof of principle for the application of U1&#95;snRNA exon skipping-based approach to correct splicing defects in RPGR .

Simple Summary: The spliceosome ribonucleoprotein complex catalyzes the removal of introns and exons ligation, a fundamental post-transcriptional process that generates mature RNAs. Cancer-associated mutations in spliceosome components give rise to aberrant splice site selection and, therefore, the production of novel isoform variants that support tumorigenesis. In this review, we summarize the current research regarding cancer hotspot mutations identified in spliceosome components acting at the very first step of splicing, namely the U1 snRNA, SF3B1, and U2AF1. Splicing alterations have been widely documented in tumors where the proliferation and dissemination of cancer cells is supported by the expression of aberrant isoform variants. Splicing is catalyzed by the spliceosome, a ribonucleoprotein complex that orchestrates the complex process of intron removal and exon ligation. In recent years, recurrent hotspot mutations in the spliceosome components U1 snRNA, SF3B1, and U2AF1 have been identified across different tumor types. Such mutations in principle are highly detrimental for cells as all three spliceosome components are crucial for accurate splice site selection: the U1 snRNA is essential for 3′ splice site recognition, and SF3B1 and U2AF1 are important for 5′ splice site selection. Nonetheless, they appear to be selected to promote specific types of cancers. Here, we review the current molecular understanding of these mutations in cancer, focusing on how they influence splice site selection and impact on cancer development. [ABSTRACT FROM AUTHOR]

The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)