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Tracy Johnson

Research

Eukaryotic genes are interrupted by stretches of noncoding DNA sequence, which are removed from the newly-synthesized pre-messenger RNA to ensure accurate expression of genetic information. In most higher eukaryotic organisms, such as humans, these noncoding sequences, introns, interrupt the majority of genes, and can be up to 100,000 bases. Hence, intron recognition is an integral step in gene expression.

Introns are excised from pre-messenger RNAs by a large ribonucleoprotein complex called the spliceosome, which is comprised of 5 small nuclear RNAs and a large collection of protein factors. The spliceosome is well-conserved from yeast to humans, and it undergoes dramatic, ATP-dependent rearrangements to allow for multiple, ordered intron recognition events and splicing catalysis. Two fundamental challenges to understanding the mechanism of pre-mRNA splicing are to characterize the dynamic RNA-RNA rearrangements that are critical for establishing the catalytic center of the spliceosome and to determine the roles of the numerous splicing proteins that are involved in this process.

Each of the steps in synthesis and processing of a messenger RNA (including pre-messenger RNA splicing) have been studied as distinct biochemical reactions. Nevertheless, there is growing evidence that in vivo, these reactions are spatially and temporally coordinated. The splicing machinery appears to associate with the pre-messenger RNA co-transcriptionally, and the transcription apparatus, including the RNA polymerase (specifically the hyperphosphorylated C-terminal domain, or CTD, of the RNA polymerase II), helps to recruit splicing factors to the nascent RNA transcript.

In order to understand these critical events in eukaryotic gene expression, we are exploiting the power of yeast genetics and biochemistry using the model organism Saccharomyces cerevisiae. S. cerevisiae is not only experimentally tractable, but its splicing machinery is very similar to that of mammals , and the genes encoding most of the splicing factors have been identified.

(1) Characterization of RNA-RNA and RNA-protein interactions involved in splice site recognition.

One of goals of the lab is to understand the dynamic rearrangements carried out by the spliceosome. In particular we have focused on the RNA-RNA and RNA-protein interactions that mediate 5' splice site recognition using a trans-splicing/crosslinking system. In vitro splicing reactions are carried out using pre-mRNAs in which the 5' splice site is contained on an RNA substituted with photoreactive, nucleoside triphosphate analogs at positions around the 5' splice site, while the 3' splice site/branchpoint are contained on a separate molecule. In this trans-splicing system, splicing proceeds through both catalytic steps to generate an accurately spliced product. Upon UV-irradiation, crosslinking is induced between the pre-mRNA substrate and the small nuclear RNAs or proteins in the reaction, allowing us to "freeze" interactions that occur between the pre-mRNA and components of the spliceosome during pre-mRNA splicing. Using this system, we have identified a number of novel interactions that take place during splice site recognition. Further, using extracts derived from yeast strains with mutations in specific splicing proteins (including the RNA helicases required for splicing), we have been able to block splicing at discreet steps along the splicing pathway, identify which proteins are responsible for mediating the observed crosslinks, and are constructing a temporal map of the interactions that occur during splicing.

(2) Identification and characterization of functional interactions between the splicing and transcription machineries.

With the growing appreciation of the close spatial and temporal relationship between transcription and splicing, it is clear that interactions between the transcription machinery and the splicing machinery play an important role in the two reactions. This has led to a model in which the transcription machinery plays a role in facilitating pre-mRNA splicing and the splicing machinery and the splicing reaction can alter the transcription properties of the polymerase (Figure 1). To explore this model, we have initiated a three-pronged approach. First we are carrying out a genetic analysis to identify specific factors that act at the interface of transcription and splicing. We are complementing this approach by using biochemical tools, including affinity purification and co-immunoprecipitation, to characterize physical interactions between these factors. Finally, we are employing a combination of in vivo and in vitro splicing and transcription assays to elucidate the mechanisms by which splicing and transcription are coordinated, including the mechanism by which pre-mRNA splicing occurs within the context of mRNA synthesis from a chromatin template.

johnson-figure Figure 1. Co-transcriptional splicing

Biography

Dr. Tracy Johnson received her Ph.D. from the University of California, Berkeley and was a Jane Coffin Childs postdoctoral fellow at the California Institute of Technology.