Control of expression of human snRNA genes

In humans, protein-coding genes and most small nuclear (sn)RNA genes are transcribed by RNA polymerase II (pol II).The carboxy-terminal domain (CTD) of the largest subunit of pol II possesses multiple heptapetide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Phosphorylation of Ser2, Ser5 and Ser7 mediates the recruitment of transcription and RNA processing factors during the transcription cycle. There are notable differences between snRNA genes and protein-coding genes in terms of mechanisms controlling their expression. Pol II does not appear to make the transition to long-range productive elongation during transcription of snRNA genes, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. Initiation of transcription of most human genes transcribed by pol II requires the formation of a preinitiation complex (PIC) comprising TFIIA, B, D, E, F and H and pol II. The general transcription factor, TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors (TAFs). Differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. It has already been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5, has been shown to be associated with pol II-transcribed snRNA genes, the full complement of TAFs associated with these genes remained unclear. Here I show, using a ChIP and siRNA-mediated knockdown approach, that the TBP/TAF complex on snRNA genes differs from that on protein-coding genes. Interestingly, the largest TAF, TAF1 and the core TAFs, TAF10 and TAF4 are not detected on snRNA genes. I propose that this snRNA gene-specific TAF subset plays a key role in gene-type-specific control of expression. In addition, in order to further understand the molecular mechanism underlying the differences between expression of protein-coding genes and snRNA genes, I have investigated the role of RNA pol II-associated protein 2 (RPAP2) in transcription of snRNA genes. Here I show that RPAP2 recognizes the phospho-Ser7 mark on the pol II CTD, siRNA mediated knockdown of RPAP2 causes defects in snRNA gene expression and that RPAP2 is a CTD Ser5 phosphatase. I also present my studies of the mechanism of inhibition of phospho-Ser2 by herpes simplex virus-1 (HSV-1) protein ICP22. Phosphorylation of Ser2 by the positive transcription elongation factor (P-TEFb) is associated with productive transcriptional elongation. However, P-TEFb is not required for elongation of transcription of snRNA genes, but functions only to activate 3' box-directed RNA processing. In addition, there are conflicting data as to whether Cdk9 is acting as a Ser2 kinase during transcription of pol II-transcribed snRNA genes. As ICP22 is thought to inhibit P-TEFb, this protein could provide an alternative means to study P-TEFb function in expression of snRNA genes.

Keywords: ICP22
Ssu72
snRNA
P-TEFb
RPAP2
TAF
HSV-1
Advisor: Murphy, S