Arrested yeast splicing complexes indicate stepwise snRNP recruitment during in vivo spliceosome assembly

Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular machine dedicated to intron removal and exon ligation. Despite an abundance of in vitro information and a small number of in vivo studies, the pathway of yeast (Saccharomyces cerevisiae) in vivo spliceosome assembly remains uncertain. To address this situation, we combined in vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation (ChIP) analysis of other splicing snRNPs along an intron-containing gene. The data indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway: first U1, then U2, and finally the U4/U6*U5 tri-snRNP. Tandem affinity purification (TAP) using a U2 snRNP-tagged protein allowed the characterization of in vivo assembled higher-order splicing complexes. Consistent with an independent snRNP assembly pathway, we observed high levels of U1-U2 prespliceosomes under U5-depletion conditions, and we observed significant levels of a U2/U5/U6/Prp19-complex mature splicing complex under wild-type conditions. These complexes have implications for the steady-state distribution of snRNPs within nuclei and also reinforce the stepwise recruitment of U1, U2, and the tri-snRNP during in vivo spliceosome assembly.     

A novel role of U1 snRNP: Splice site selection from a distance

Removal of introns by pre-mRNA splicing is fundamental to gene function in eukaryotes. However, understanding the mechanism by which exon-intron boundaries are defined remains a challenging endeavor. Published reports support that the recruitment of U1 snRNP at the 5′ss marked by GU dinucleotides defines the 5′ss as well as facilitates 3′ss recognition through cross-exon interactions. However, exceptions to this rule exist as U1 snRNP recruited away from the 5′ss retains the capability to define the splice site, where the cleavage takes place. Independent reports employing exon 7 of Survival Motor Neuron (SMN) genes suggest a long-distance effect of U1 snRNP on splice site selection upon U1 snRNP recruitment at target sequences with or without GU dinucleotides. These findings underscore that sequences distinct from the 5′ss may also impact exon definition if U1 snRNP is recruited to them through partial complementarity with the U1 snRNA. In this review we discuss the expanded role of U1 snRNP in splice-site selection due to U1 ability to be recruited at more sites than predicted solely based on GU dinucleotides.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

Aberrant splicing of tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism linked to chromosome 17

Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.     

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single‐molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1‐independent process of binding to an ESE. Structural analysis and cross‐linking data show that SRSF1 contacts U1 snRNA stem‐loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.     

Ser/Arg-rich protein-mediated communication between U1 and U2 small nuclear ribonucleoprotein particles

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.     

Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL,the U1 snRNP and U2AF underlie male exon skipping

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANS-FILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.     

RBFOX2 promotes protein 4.1R exon 16 selection via U1 snRNP recruitment

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5' splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5' splice site, but not the engineered strong 5' splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5' splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5' splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5' splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.     

Functional studies on the ATM intronic splicing processing element

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ~40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.