Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components.

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

Characterization of the aberrant splicing of MAP3K7 induced by cancer-associated SF3B1 mutation

SF3B1, an essential RNA splicing factor, is frequently mutated in various types of cancers, and the cancer-associated SF3B1 mutation causes aberrant RNA splicing. The aberrant splicing of several transcripts, including MAP3K7, promotes tumorigenesis. Here, we identify a premature termination codon in the aberrantly spliced transcript of MAP3K7. Treatment of HEK293T cells transfected with the K700E-mutated SF3B1 with cycloheximide leads to increased accumulation of the aberrant spliced transcript of MAP3K7, demonstrating that the aberrantly spliced transcript of MAP3K7 is targeted by nonsense-mediated decay. The aberrantly spliced MAP3K7 transcript uses an aberrant 3’ splice sites and an alternative branchpoint sequence. In addition, the aberrant splicing of MAP3K7 requires not only the polypyrimidine tract associated with normal splicing but also an alternative polypyrimidine tract upstream of the aberrant 3’ splice site. Other cancer-associated SF3B1 mutations also cause the aberrant splicing of MAP3K7, which depends on the same sequence features. Our data provide a further understanding of the mechanisms underlying aberrant splicing induced by cancer-associated SF3B1 mutation, and reveal an important role of alternative polypyrimidine tract in diseases.

Spliceosome: Physiology and Disease Pathogenesis

Pre-mRNA splicing is an essential step for the expression of intron-containing genes, i.e., the majority of genes. Splicing is a high-fidelity process, as required for the correct expression of proteins. However, there is flexibility in the selection of competing splice sites, which gives rise to alternative splicing, a regulated process that greatly increases the diversity of the proteome. Splicing is catalyzed in a stepwise manner by the spliceosome, a macromolecular machine that consists of 5 small RNAs and more than 100 proteins. Key insights about the structure and dynamics of spliceosomal complexes have recently been obtained through cryo-electron microscopy studies. Dysregulated splicing can arise from mutations in the splice sites or regulatory elements of individual genes, from alterations in the levels of regulatory splicing factors (activators and repressors), or from mutations in splicing-factor genes. All of these scenarios can give rise to various cancers, depending on the affected gene and the cellular context. Interestingly, recurrent somatic heterozygous mutations in particular splicing factors involved in branchpoint-sequence and 3'-splice-site recognition have emerged as key drivers of certain myeloid neoplasias. This presentation will review relevant features of the spliceosome machinery, the functional implications for normal and pathological conditions, and the potential for novel therapies. Disclosures Krainer: Ionis Pharmaceuticals: Consultancy, Honoraria, Patents & Royalties, Research Funding; Stoke Therapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties; Cold Spring Harbor Laboratory: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; H3 Biomedicine: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Construction of a Herpes Simplex Virus Type 1 Mutant with Only a Three-Nucleotide Change in the Branchpoint Region of the Latency-Associated Transcript (LAT) and the Stability of Its Two-Kilobase LAT Intron

ABSTRACT Previous studies using a eukaryotic expression system indicated that the unusual stability of the latency-associated transcript (LAT) intron was due to its nonconsensus branchpoint sequence (T.-T Wu, Y.-H. Su, T. M. Block, and J. M. Taylor, Virology, 243:140-149, 1998). The present study investigated the role of the branchpoint sequence in the stability of the intron expressed from the herpes simplex virus type 1 (HSV-1) genome and the role of LAT intron stability in the HSV-1 life cycle. A branchpoint mutant called Sy2 and the corresponding rescued viruses, SyRA and SyRB, were constructed. To preserve the coding sequence of the immediate early gene icp0, which overlaps with the branchpoint region of the 2-kb LAT, a 3-nucleotide mutation into the branchpoint region of the 2-kb LAT was introduced, resulting in a branchpoint that is 85% identical to the consensus intron branchpoint sequence of eukaryotic cells. As anticipated, there was a 90- to 96-fold reduction in 2-kb LAT accumulation following productive infection in tissue culture and latent infection in mice with Sy2, as determined by Northern blot analysis. These results clearly suggest that the accumulation of the 2-kb intron in tissue culture and in vivo is, at least in part, due to the nonconsensus branchpoint sequence of the LAT intron. Interestingly, a failure to accumulate LAT was associated with greater progeny production of Sy2 at a low multiplicity of infection (0.01) in tissue culture, but not in mice. However, the ability of mutant Sy2 to reactivate from trigeminal ganglia (TG) derived from latently infected mice was indistinguishable from that of wild-type virus, as assayed in the mouse TG explant reactivation system.

A Potential Role for U2AF-SAP 155 Interactions in Recruiting U2 snRNP to the Branch Site

ABSTRACT Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3′ splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155’s downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.