IV. Mechanisms of Complex

Splicing: NTC/Prp19C has a critical role in its involvement as a tool used for splicing. Splicing removes introns and keeps exons used in the final RNA product. What Prp19C does for the splicing reaction is it’s regulation of the formation and progression of essential spliceosome conformations. Prp19C allows for proper splicesome conformation needed in order for the splicosome to bind to RNA and properly remove introns. Specifically, NTC/Prp19C is a non-snRNP splicing complex used in multiple steps involving splicing. NTC/Prp19C associates with the assembling spliceosome during or after the dissociation of the U4 snRNP, stabilizes the U5/U6 snRNP in the activated spliceosomal complex (Bact) and remains associated with the spliceosome during the second step of splicing.

Genome maintenance: Prp19 was also identified in screens for mutations that lead to increased sensitivity to DNA damage from UV radiation, free radicals and carcinogenic reagents and a direct function of NTC/Prp19C in genome maintenance. Prp19 was first identified in S. cerevisiae in a genetic screen for mutations conferring sensitivity to the DNA damaging reagent psoralen as well as other interstrand cross-link (ICL) inducing reagents and termed Pso4. In the human homolog, Prp19 was purified from human cell extracts used as a component of the nuclear matrix and named NMP200. Researchers found that NMP200/hPRP19 interacts with the terminal deoxynucleotidyl transferase (TdT) and is involved in mediating cell survival after DNA damage.Also the loss of hPso4/hPRP19 resulted in accumulation of double-strand breaks (DSBs). Not only that but the human Pso4 also interacts with metnase, a protein which contributes to DSB repair and restart of stalled replication forks, and is necessary to bring metnase to DSB sites which is a result of its Pso4/PRP19 structure.

Degradation of proteins by the proteasome: NTC/Prp19C has a direct function in the degradation of proteins by the proteasome. Prp19 contains a U-box domain at its N-terminus, which is structurally related to the RING finger motif found in certain E3 ubiquitin ligases, and exhibits E3 ligase activity in vitro and in vivo. Prp19 also participates in the canonical ubiquitin–proteasome pathway as many E3 ligases do due to similarities in its structure and function to that of a E3 ligase that is used in protein degradation by the proteasome. Research with mouse Prp19 in vivo and in vitro interacts with mouse SUG1 (mSUG1) which is a subunit of 19S regulatory part of the proteasome. The inhibition of the proteasome led to accumulation of mPrp19 with mSUG1 in the cytoplasm. It was also found that the activity of the proteasome was increased in cells overexpressing mPrp19 linking the mPrp18 to proteasome expression and protein degradation. Also human PRP19 as well as its yeast counterpart interact with PSMB4/Pre4/beta7 which is a component of the 20S catalytic core of the proteasome that functions in recruitment of ubiquitylated proteins to the proteasome for degradation. These results show that Prp19 functions in protein degradation and also aids in the transportation of its substrates to the proteasome by binding to proteasomal subunits.

Transcription elongation: NTC/Prp19C functions in transcription elongation and as a result is important for two steps of gene expression. In yeast NTC/Prp19C functions in transcription elongation by enabling the binding of the TREX complex at transcribed genes. TREX is a protein complex coupling transcription to mRNA export. In yeast, TREX consists of the subcomplex THO (comprised of Tho2, Hpr1, Mft1, Thp2 and Tex1), the mRNA export factors Sub2 and Yra1 and the mRNA-binding proteins Gbp2 and Hrb1. TREX is recruited to transcribed genes and moves along genes together with the transcription machinery. It is binded co-transcriptionally to the synthesized mRNA which inhibits the R-loops formed by RNA–DNA hybrids leading to hyper-recombination. TREX functions in co-transcriptional assembly of the mRNA into an mRNP. NTC/Prp19C interacts with TREX as well as RNAPII and is recruited to intron-containing and intronless genes. In cells expressing a C-terminal truncated Syf1 NTC/Prp19C still interacts with TREX, but its interaction with RNAPII and its recruitment to genes is reduced greatly as a result meanwhile TREX occupancy at genes is decreased. The lower TREX levels at genes is the source for the transcription defect observed in the syf1 mutant. Since TREX occupancy in the syf1 mutant mainly decreases at the 3? end of genes NTC/Prp19C most likely serves the role of stabilizing TREX at genes rather than recruiting it. In short, yeast NTC/Prp19C functions in transcription elongation by ensuring the stabilization of TREX.