I am very grateful to be a member of the Patton Lab, where I have worked with so many wonderful people. I would like to thank all Patton Lab members, past and present, for providing a warm, happy and friendly environment that makes life in the lab easier and truly enjoyable. I would especially like to thank Daron Barnard, Billy Dye, Jun Li, Izzy Pérez, and Brooke Thompson for their directness.
Jun Li, my best friend inside and outside the lab, thanks again for many helpful discussions, as well as for all the others. I would also like to thank the administrative staff of the Department of Biological Sciences, especially Roz Johnson and Nancy Jackson, for their dedicated assistance over the years. The 5' and 3' splice sites define the exon/intron boundaries, while the branch point provides an adenosine for nucleophilic attack at the 5' splice site.
Coupling occurs in two consecutive transesterification reactions, the first of which involves 5' cleavage. Although the basic chemistry of splicing is remarkably conserved and quite simple, splicing is indeed a very complex task given the small size of the cis-elements (splice sites and branch point) and the size of the eukaryotic genome.
Exon2 Exon1
The consensus sequences for higher eukaryotic 5' splice site (5' SS), 3' splice site (3' SS), branch point (BP) and polypyrimidine tract (Py) are indicated (N = any nucleotide, Y = pyrimidine, R = purine).
3’ SSBP
Exon2Exon2
Exon1
The model adapted from Stevens et al. 2002 ) showing the yeast penta-snRNP assembled on the pre-mRNA substrate as a preformed particle. Strikingly, the penta-snRNP is able to cleave when supplied with micrococcal nuclease-treated nuclear extracts and thus functions as a preformed particle (Stevens et al., 2002). Despite the two possibly conflicting models of spliceosome assembly, it has been agreed for years that the spliceosome is a large, complex particle (Moore et al., 1993).
When coupled with NMD, about 18–25% of the alternatively spliced exons are predicted to regulate transcript abundance (reviewed in Stamm et al., 2005). Alternative splicing is regulated through the combinatorial interplay between cis-acting elements of variable strength and the binding of trans-acting proteins (reviewed in Matlin et al., 2005; Smith and Valcarcel, 2000). Recently, another DBHS domain containing protein, Paraspeckle Protein 1 (PSP1), was identified in humans (Andersen et al., 2002; Fox et al., 2002).
Immunodepletion of PSF from HeLa nuclear extract first suggested that PSF may play a role in early spliceosome formation ( Patton et al., 1993 ), but subsequently. Furthermore, PSF has been identified as part of a snRNP-free U1A complex, which has a potential role in both splicing and polyadenylation (Lutz et al., 1998).
Pre-mRNA splicing
Nuclear RNA retention/export
Topoisomerase activity
PSFPSF
These results suggest that in the absence of the original stem 1b structure (compare the 5' mutant with the 3' mutant), the wild-type sequence in the 3'. However, in the presence of the original stem structure, the stem sequence is apparently less important for binding specificity (compare the 5'-3' mutant with wild-type U5). Thus, PSF and p54nrb appear to function at multiple steps of the splicing pathway, one of which may be
A speculative possibility is that PSF and p54nrb could bind U5 in two ways: to the intact stalk or to the 3' side of the stalk after melting. First, PSF and p54nrb may initially bind to double-stranded stem 1b, but then bind more closely to the 3' side of the stem upon unwinding. The PCC complex contains all five splicing snRNPs and is close in size to the spliceosome.
Interestingly, neither exogenous pre-mRNA nor ATP hydrolysis is required for PCC complex formation. 30 µl of HeLa nuclear extracts (approximately 150 µg) were adapted to conjugation conditions (see above) to allow PCC formation. Data processing of SEQUEST output files was performed as previously described (Link et al., 1999).
HeLa nuclear extracts adapted to splicing conditions with or without addition of radiolabeled AdML pre-mRNA were incubated at 30°C for 15 min to allow formation of the SCC. All five splicing snRNPs coimmunoprecipitate with PSF in HeLa nuclear extracts adapted to splicing conditions without addition of pre-mRNA. We then used immunoprecipitation experiments to further analyze the formation of the PCC complex in the presence or absence of pre-mRNA and at different time points.
Formation of the PCC complex in depleted nuclear extracts was then analyzed by IP using PSF antibodies under splicing conditions. Three years ago, the discovery of the yeast penta-snRNP suggested that not all spliceosomes assemble de novo in a stepwise fashion (Stevens et al., 2002). PSF-p54nrb now binds the 3' side of the stem and helps stabilize the unfolded U5 structure.
First, assembly of active spliceosomes requires ATP hydrolysis whereas neither ATP nor exogenous pre-mRNA is required for PCC formation. Identification by mass spectrometry and functional analysis of novel yeast [U4/U6.U5] tri-snRNP proteins. Proximity of the invariant loop of U5 snRNA to the second intron residue during pre-mRNA splicing.
Functional analysis of U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing.
