Question

The SR protein SRn2 can auto-regulate its own level using a type of alternative splicing known as "unproductive splicing." The SRn2 gene (shown above) can be alternatively spliced to give two distinct mRNAs: mRNA (A) is stable and actively translated, while mRNA (B) includes the alternative exon 2a and is targeted by nonsense mediated decay (NMD). It is observed that when levels of SRn2 protein rise, the levels of its productively spliced mRNA fall (and vice versa).

SRN2 gene: transcription start AUG stop 1 2 2a 3 4 SRN2 protein: SRN2 MRNAS: actively translated SR AAА 3' (A) 5' m7G subject to NMD ААА 3' (B) 5' 7mG 5' m7G AAA 3'

1) Propose a model for the autoregulation of the SRn2 pre-mRNA by SRn2 protein. Your model should provide a specific mechanism for how the SR protein regulates splicing of its pre-mRNA, and provide an explanation for why mRNA (B) is a target of NMD. [12 points]

Answer 1

This experiment suggest that introns within SRn2 gene governs the autoregulation of SR protein expression. Some structural elements within intronic SRn2 gene were found to be important for splicing efficiency. The regulation of expression of SRn2 gene is dependent upon extra-ribosomal autoregulatory functions of the SR proteins themselves. Here SR proteins have been found to regulate the splicing of their own pre-mRNA transcripts. Model of autoregulation is of structural basis. Such direct regulation can be characterized for the SR protein, which binds to an RNA secondary structure in the precursor transcript and inhibits its splicing. Similarly, the SR protein self-regulates its expression by directly binding to a stem loop structure in the SRn2 pre-mRNA. Pre-mRNA of SRn2 gene contains an intronic alternative 5’ splice site and splicing at this site gives rise to a transcript that is degraded by the cytoplasmic nonsense-mediated decay (NMD) pathway. Also, the inhibition of splicing is specific to the annotated splice site. The introns within SRn2 gene are also capable of regulating both inter- and intragenic expression.

SR protein regulates splicing of its pre-mRNA by structural elements within intronic SRn2 gene. To simplify the process of manipulating the intron, we shall first construct a plasmid-based reporter in which the SRn2 gene intron was attached to a transcript encoding green fluorescent protein (GFP). We shall then utilized site-directed mutagenesis to sequentially bifurcate the intron and assessed loss of splicing inhibition by comparing relative levels of spliced and unspliced reporter transcript by Northern blot. Through this approach, we will isolate a region spanned by intronic nucleotides that harbors a prominent secondary RNA structure and that are necessary to confer splicing autoregulation. This structure consists of a series of base-paired stems and internal loops closed by an apical hairpin loop. Thus leading to nonsense-mediated decay (NMD) pathway.