Engineered pauses alters splicing patterns

For my postdoctoral research, I accepted a position in the lab of Dr. Manual Ares Jr. to focus on two major molecular pathways: transcription and splicing. The majority of mRNAs are spliced as they are made by RNA polymerase II (RNAPII), but how these two processes are co-regulated is still somewhat of a mystery. In collaboration with Haller Igel and Dr. Karla Neugebauer’s’ group at Yale, we set out to understand the connection between transcription rate and downstream effects on splicing patterns.  Our approach was to design a 21-nt DNA element (Artificial Arrest or AAR) that we predicted would pause RNAPII during transcription. If we could engineer a transcriptional pause, we might have a chance of figuring out the feedback to the splicing pathway. I discovered that our pause element did indeed cause RNAPII to slow down during transcription with a series of biochemical experiments. My collaborator Haller Igel discovered that in the model organism S. cerevisiae the AAR promoted a change in splicing patterns were changes depended on the position of the AAR in a minigene. With the help of the Neugebauer lab we learned the single molecule technique Single Molecule Intron Tracking (SMIT) and were able to show that we could detect a pause in living yeast cells that mirrored the pause we detected in vitro. This work demonstrates a connection between transcription rate and splicing that is position-dependent and seems to be linked to early splicing assembly. Our manuscript is in preparation.