Spinal Muscular Atrophy (SMA) is an autosomal recessive disease that leads to specific loss of motor neurons. It is caused by deletions or mutations of the survival of motor neuron 1 gene (SMN1). The remaining copy of the gene, SMN2, generates only low levels of the SMN protein due to a mutation in SMN2 exon 7 that leads to exon skipping.

To correct SMN2 splicing, we use Adenovirus type 5–derived vectors to express SMN2-antisense U7 snRNA oligonucleotides targeting the SMN intron 7/exon 8 junction. Infection of SMA type I–derived patient fibroblasts with these vectors resulted in increased levels of exon 7 inclusion, upregulating the expression of SMN to similar levels as in non–SMA control cells.

These results show that Adenovirus type 5–derived vectors delivering U7 antisense oligonucleotides can efficiently restore full-length SMN protein and suggest that the viral vector-mediated oligonucleotide application may be a suitable therapeutic approach to counteract SMA.

Eukaryotic gene expression is coordinated through a series of processes from which RNA is transcribed, processed, and translated into the proteins that serve as the functional building blocks of complex cellular organisms. These steps are highly integrated, often occurring in the same spatial and temporal space. Although this co-transcriptional connection is well described, it remains unclear how the concerted rates of global RNA processing steps affect the final mRNA isoform. Here we disrupt steady-state RNA levels using 4-thiouridine (4sU) metabolic labeling and utilize high-throughput sequencing to determine the global rates of pre-mRNA processing. We find that introns that display higher retention are subject to slower splicing kinetics, with longer introns being removed quicker. Exon skipping is subject to competing splice site pairing kinetics and size constraints that highlight optimal exon recognition features by the spliceosome. Integration of this information permits the determination of the order of intron removal across entire genes, thus producing detailed gene RNA processing maps.

Intron retention and exon skipping (cassette exons) are two types of alternative splicing that can be regulated by SR proteins by strengthening the recognition of introns and exons that are otherwise prone to alternative splicing. To test the hypothesis that SR proteins modulate alternative splicing through changes in splicing kinetics, we depleted SRSF1 in human hepatocellular carcinoma cells and derived RNA processing rates. Loss of SRSF1 leads to higher intron retention and more exon skipping. This is primarily achieved through changes in splicing rates and Pol II density, with a strong dependence on optimal feature length constraints. eCLIP data further demonstrates that SRSF1 binds preferentially to weaker exons that are prone to being skipped.

Together these data suggest that alternatively spliced introns and exons have distinct kinetic profiles, constrained by lengths that favor exon definition. The splicing factor SRSF1 acts primarily as an activator, promoting the constitutive splicing of exons and introns through the modulation of Pol II density and subsequent splicing kinetics.

The eukaryotic genome is a large and complex network of molecules that work together to create diversity across many different species. Pre-mRNA splicing is a vital step in the processing of RNA into functionally diverse proteins. Splicing has a vast history and understanding the combination of many different factors and elements is fundamental to studying gene expression.

Gene evolution and the evolutionary pressures that encode a set of exonic sequences maintain efficient pre-mRNA splicing and ultimately dictate the selection of amino acids that define a protein. Exonic sequences have been regulated in a way to demonstrate the co-existence of coding and splicing pressures. Through the design of an exon conservation database, evolutionary conservation patterns were identified that influenced the final sequence of an exon. This information led to important predictions about splicing patterns in human disease. The database allowed for the identification of essential architectural parameters of the human genome. In addition, analysis of nucleotide variations at the wobble position identified splice altering SNPs and how these SNPs influenced exon inclusion.

Regulation of alternatively spliced exons requires a coordinated effort by many cis and trans-acting factors. Understanding how these factors work together is important for the mechanism of alternative splicing regulation. SR proteins and hnRNPs have previously demonstrated a position-dependent method of regulation. It was shown that U1 snRNP, at activating or repressive conditions, displayed dynamic changes in its compositional integrity. This demonstrated that U1 snRNP integrity is therefore modulated by the presence of position-dependent interactions with splicing regulatory factors, further suggestive of U1 snRNP as a molecular gatekeeper for splicing initiation.

Polyadenylation is a fundamental step in the 3’ end processing of mRNA. Alternative polyadenylation is another contributor to genomic diversity. The coordinated efforts between splicing and polyadenylation have been demonstrated for terminal exons, however, understanding the influence these two processes have on upstream exons was unknown. Genome-wide analyses allowed for the identification of an important role that a polyadenylation factor (CstF64) has on alternative splicing. In addition, the coupling that was seen between alternative polyadenylation and alternative splicing was in fact limited to terminal exons.

The process of pre-mRNA splicing is conserved from yeast to humans. Alternative splicing drives transcriptomic and proteomic diversity in higher eukaryotes through the creation of multiple unique mRNA transcripts from one gene encoded pre-mRNA. Alternative splicing is under combinatorial control meaning that many cis-acting sequence elements and trans-acting protein factors affect splice site usage. A thorough understanding of the factors that drive alternative splicing is essential in order to understand normal and aberrant splicing in the cell. Splicing regulatory proteins often drive tissue specific alternative splicing. The two most well characterized classes of splicing regulatory proteins are the SR proteins and hnRNPs. These two classes of proteins were shown to have a position-dependent activity on splicing efficiency when bound upstream or downstream of a regulated 5' splice site. To test the hypothesis that splicing regulators affect splicing efficiency by altering the structure of U1 snRNA, a pulldown approach was designed to isolate native U1 snRNPs bound to activated or repressed E-complexes. SHAPE chemistry and psoralen crosslinking were used to show that the core of U1 snRNA does not undergo structural rearrangements, however the 5' end of U1 snRNA is more base paired to the 5' splice site when splicing activators are bound. This study helped to elucidate the mechanism of position-dependent splicing activation and repression. Splice site strength and intron-exon architecture are the primary drivers of the innate ability of an exon to undergo alternative splicing. Previous work had shown that the proximity of the 5' and 3' splice site dictate splice site usage under intron definition. It was hypothesized that the proximity of the 5' and 3' splice sites across the exon influence splice site usage under exon definition. A data base of alternative splicing events comprised of exons with two competing 5' splice sites and one constant 3' splice site was created. Filtering of data to account for differences in MaxEnt scores (computational scores that predict 5' and 3' splice site usage) showed that a genome-wide exon centric proximity rule exists. Observations from the computational studies were confirmed with in vivo minigene analysis. This thesis expands upon the molecular mechanisms of splice site selection in the context of combinatorial alternative splicing. The findings presented herein will aid in the understanding of normal and aberrant alternative splicing programs.