Ohi and Gould laboratories to critically evaluate my work, our scientific collaborations and friendships over the years. You were a scientific inspiration and taught me many valuable life lessons, which I will cherish forever. To Mel, thank you for everything you have done for me over the years.
You have provided me with so many opportunities through specialized training and collaborations, which have furthered my scientific education. Although the laboratory has changed over the years, you have always kept the laboratory environment a positive and friendly place to work and for that I will always be grateful. Thank you for joining me in my long hypothetical discussions about matters loosely related to science.
Finally and most importantly, I would like to thank my friends and family who have provided encouragement and faith over the years. To my wife Sarah Collier: “You are fire, you are dynamite” and without you I could not have made it through this journey.
Introduction
The general location of the 5' and 3'SS (splice site) and BP (split point) is indicated. The 5' end of the pre-mRNA is capped with a 7-methylguanosine (m7G) nucleotide, which protects the RNA from. Recognition sequences are found at the 5' end of the intron (GUGYG, Y is any pyrimidine) and at the splice site (AY) (Saldanha, Mohr et al. 1993).
Comparison of active site RNA between Group II Introns and the Spliceo- model (A) of U2/U6 snRNA in the active site of the spliceosome (Burke et al. 2012; PDB 2LKR). The NTC is conserved from yeast to humans and is a single protein subcomplex of the spliceosome named after its founding member Sc Prp19 (Tarn, Hsu et al. 1994). NTC also activates the spliceosome through promoting the stable interaction of U5 and U6 snRNAs with the 5'SS (Chan and Cheng 2005).
Sc Cwc2 (homolog of Sp Cwf2) interacts with both snRNA and pre-mRNA in vitro, and the RRM can bind RNA independently of the Zn finger, but to a reduced level (McGrail, Krause et al. 2009). The RNA helicases are members of the superfamily2 (SF2) group of RNA helicases, which are typically monomeric with two RecA-like domains flanked by variable N- and C-terminal extensions (Cordin and Beggs 2013) (Fig. 1- 6B). In humans, Hs DDX39B (Sp Uap56) uses its helicase activity to remove Hs U2AF65 (Sp Prp2), allowing the U2 snRNP to bind tightly, leading to the formation of the A complex (Fleckner, Zhang et al. 1997 ).
In coordination with Sp Uap56, Sp Prp11 works directly on the U2 snRNP to promote the formation of the A complex (Xu, Newnham et al. 2004). Activation of the spliceosome begins with the recruitment of Sp Cdc28 to the Bact complex (Fig. 1-3B). Stable integrants were selected based on resistance to 5-fluoroorotic acid (5- FOA) (Livesay, Collier et al. 2013) and acquisition of the LEU- phenotype.
Eight conserved RNA helicases (Sp Uap56, Sp Prp11, Sp Prp28, Sp Brr2, Sp Cdc28, Sp Prp16, Sp Prp22 and Sp Prp43) drive the spliceosome transitions between different stages of the splicing cycle. The essential nature of the N-terminus of Sc Prp16 is due to its role in recruiting Prp16 to the spliceosome (Wang, Wagner et al. 1998). Since the CS phenotype of Sp prp16-1 has been previously characterized (Urushiyama, Tani et al. 1996), we began to investigate the HS phenotype of the Sp prp16-1 mutant.
Using RT-PCR we analyzed the state of the cwf8 pre-mRNA transcript in the Cdc5-TAP prp16-1 complex. The absence of pre-mRNA and the presence of the lariat RNA as well as mRNA suggest that the Cdc5-TAP prp16-1 complex is a second-step splicing complex. Analysis of the Cdc5-TAP prp16-1 Complex (A) Anti-Cdc5 Western blot of a linear glycerol gradient with purified ILS (Cdc5-TAP) and Cdc5-TAP prp16-1. m3.
In contrast, the presence of the SF3A and SF3B complexes may represent the P complex found in humans (Ilagan, Chalkley et al. 2013).
Discussion
34;Characterization of purified human Bact-spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first-step catalysis." RNA. 34;Genetic analysis reveals a role for the C-terminus of the Saccharomyces cerevisiae GTPase Snu114 during spliceosome activation.". 34;Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation from U4 in a similar manner to Prp19p." Mol Cell Biol.
34; "Protein Composition and Electron Microscopy Structure of Affinity Purified Human Spliceosome Complexes B Isolated under Physiological Conditions." Mol Cell Biol. 34; Dissection of Factorial Requirements for Spliceosome Disassembly and Elucidation of Its Dissociation Products Using a Purified Splicing System." Genes Dev. 34; Metal ion catalysis during the exon ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns.” RNA.
34; Function of the Nineteen Complex (NTC) in regulating spliceosome conformations and fidelity during pre-mRNA splicing." Biochem Soc Trans. 34; Pre-mRNA splicing in Schizosaccharomyces pombe: regulatory role of a kinase conserved from mammalian fission yeast. " Curr Genet. 34; A self-splicing group II intron in the mitochondrial large subunit rRNA (LSUrRNA) gene of the eukaryotic alga Scenedesmus obliquus.” Nucleic Acids Res.
34;A link between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase." 34;Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts that Myb contains.-related Cdc5p/Cef1p, novel pre-mRNA splicing factors and snRNAs." Mol Cell Biol. 34;ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA.".
34; Transcript specificity of yeast pre-mRNA splicing revealed by mutations in spliceosome core components." PLoS Biol 5(4): e90. 34; Yeast precursor mRNA processing protein PRP19 associates with the spliceosome concurrently with or immediately after dissociation of the small core U4 RNA." Proc Natl Acad Sci USA. 34; One gene, many proteins: alternative splicing of the ryanodine receptor gene adds novel functions to an already complex channel protein.” Circ Res.
34;Functional contacts with a series of splicing proteins suggest a central role for Brr2p in the dynamic control of the sequence of events in spliceosomes of Saccharomyces cerevisiae. .” Gene Dev.
