2008 Rustbelt RNA Meeting






Talk abstracts

Talk on Friday 04:30-04:50pm submitted by Caroline Lee

The ability to catalyze protein-free splicing is phylogenetically conserved from yeast to human

Caroline Lee (Biochemistry, Case Western Reserve University), Saba Valadkhan (Biochemistry, Case Western Reserve University)

We previously showed that protein-free fragments of human U6 and U2 snRNAs performed a two-step splicing reaction on short RNA substrates. To determine if the ability to catalyze splicing without the help of proteins is a phylogenetically conserved feature of the spliceosomal snRNAs, we performed a detailed analysis of the structural and catalytic features of U6 and U2 snRNAs derived from human and yeast. The genetically deduced secondary structure of the yeast U6/U2 complex is different from that in human. Also, yeast U6 snRNA shows divergence from the consensus sequence in a number of residues of the catalytically critical central domain. While the RNAstructure algorithm predicts a similar global base-paired structure for human and yeast U6/U2 complexes, a number of structural elements, such as U6/U2 helices I and III, and the U2 stem I are predicted to fold differently.
In order to study the catalytic properties of the yeast snRNAs, we first determined if yeast U6 and U2 can perform protein-free splicing. Although the snRNAs could bind the splicing substrates, they did not show catalytic activity under any of the conditions tested, while human U6/U2 was active. To determine whether this resulted from the differences in sequence or secondary structure of the yeast and human snRNAs, we made small, directed mutations in yeast U6 and U2. These mutations induced individual domains of the yeast U6/U2 complex to form basepairing interactions similar to those predicted to form in human by RNAstructure algorithm and genetic analyses. A triple point mutation in the helix II region was introduced to stabilize this helix. Two point mutations in helices I and III induced these two helices to form a structure similar to that predicted to form in human. Interestingly, while introduction of the helix II or helix III mutations did not result in catalytic activity, modification of helix I led to a weak activity in protein-free splicing assays. A triple mutant that contained the helix II and III mutations in addition to the helix I point mutation showed enhanced activity. These results underscore the crucial contribution of the secondary structure of the U6/U2 complex to catalytic function and further indicate that the ability to perform protein-free splicing is phylogenetically conserved from yeast to human.

Keywords: U6U2, Splicing, Catalysis