U1 snRNA Variants Research Articles

Examining the capacity of human U1 snRNA variants to facilitate pre-mRNA splicing.

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.

Human spliceosomal snRNA sequence variants generate variant spliceosomes.

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type–specific alternative splicing patterns.

An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency.

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5′ splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A.

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5’ splice site (5’ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5’ss is surrounded by various cryptic 5’ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5’ss and create/strengthen cryptic 5’ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5’ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5’ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5’ss (∼80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5’ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A.

Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism.Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATRwt context), because the change was ineffective when the weak 5′ or the 3′ splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS.A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5’ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice.Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome.

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5′ splice sites at exon/intron boundaries. U1 snRNAs associate with 5′ splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the ‘Smith antigen’, or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs.

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

U1-like snRNAs lacking complementarity to canonical 5′ splice sites

We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5' splice site (5'SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA-protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes.

U1 snRNA variants coexist in Bombyx mori cells

Evidence for at least four U1 snRNA variants were obtained from a U1 cDNA library using U1 snRNA from Bombyx mori BmN cells in culture. Sequence analysis of thirty cDNA clones showed that: (1) the nucleotide changes are in the hairpin structures I, II and III; (2) the majority of the base changes in stem structures between a posterior silk gland (PSG) U1 RNA and the BmN U1 clones, as well as among the BmN U1 clones, are compensatory; (3) although the base differences between PSG U1 and BmN U1 clones, and among the BmN U1 clones, are not the same, they are located in similar positions in moderately conserved sites, frequently at the bases of loops; (4) when comparing the PSG U1 with the BmN U1 clones, twelve out of nineteen stem differences generate stronger pairing resulting in a more stable hairpin II in the BmN U1 clones; and (5) the Sm and 70K proteins binding site sequences are highly conserved among these U1 clones. Although a comparison of sequences changes associated with U1 isoforms from different species indicate that there are no common base changes with the B. mori U1 clones reported here, similarities in the multitude and location of base differences in hairpins I, II and III are observed in mouse and/or Xenopus. It is possible that U1 variants like the ones reported here play a role in alternative pre-mRNA splicing by way of different RNA-protein factor interactions.

Developmental expression of plant snRNAs

Although the number of plant U1, U2, U4 and U5 small nuclear RNA (snRNA) variants sequenced has steadily increased over the past few years, the function of these variants in plant splicing is still not understood. In an effort to elucidate the function of plant snRNA variants, we have examined the expression of U1-U6 snRNA variants during pea seedling development. In contrast to mammalian nuclei which express a single, abundant form of each snRNA, pea nuclei express several equally abundant variants of the same snRNA. Comparison of the snRNAs in pea seeds and seedlings has revealed that four (U1, U2, U4, U5) of the five snRNAs required for pre-mRNA splicing have differentially- and developmentally-regulated forms detectable on Northerns. Only U6 snRNA, which fractionates as a single species on Northerns, appears to be constitutively expressed. Switches in the expression of the pea U1, U2 and U4 snRNAs occur at three distinct stages in development: seed maturation, seed germination and seedling maturation. Surprisingly, the snRNA profiles of mature desiccated seeds and mature leaf tissues are nearly identical and different from developing seeds and seedlings suggesting that switches in the snRNA population occur at transitions between active and inactive transcription. Sequence analysis and differential hybridization of the U1 snRNA variants has demonstrated that some of the developmentally-regulated forms represent sequence variants. We conclude that select subsets of pea snRNAs accumulate at particular stages during plant development.

Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants

The interaction of the U1-specific proteins 70k, A and C with U1 snRNP was studied by depleting gradually U1 snRNPs of the U1-specific proteins by Mono-Q chromatography at elevated temperatures (20-37 degrees C). U1 snRNP species were obtained which were selectively depleted of either protein C, A, C and A, or of all three U1-specific proteins C, A and 70k while retaining the common proteins B' to G. These various types of U1 snRNP particles were used to study the differential accessibility of defined regions of U1 RNA towards nucleases V1 and S1 dependent on the U1 snRNP protein composition. The data indicate that in the U1 snRNP protein 70k interacts with stem/loop A and protein A with stem/loop B of U1 RNA. The presence or absence of protein C did not affect the nuclease digestion patterns of U1 RNA. Our results suggest further that the binding of protein A to the U1 snRNP particle should be independent of proteins 70k and C. Mouse cells contain two U1 RNA species, U1a and U1b, which differ in the structure of stem/loop B, with U1a exhibiting the same stem/loop B sequence as U1 RNA from HeLa cells. When we used Mono Q chromatography to investigate possible structural differences in the two types of U1 snRNPs, we observed that protein A was always preferentially lost from U1b snRNP as compared to U1a snRNPs. This indicates that one consequence of the structural difference between U1a and U1b is a lowering of the strength of binding of protein A to U1b snRNP. The possible functional significance of this finding is discussed with respect to the fact that U1b RNA is preferentially expressed in embryonal cells.

Drosophila melanogastergenes for U1 snRNA variants and their expression during development

We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. Using specific primer extension assays, we have observed that the U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.