An Investigation of a Role for U2 snRNP Spliceosomal Components in Regulating Transcription

There is mounting evidence to suggest that the synthesis of pre-mRNA transcripts and their subsequent splicing are coordinated events. Previous studies have implicated the mammalian spliceosomal U2 snRNP as having a novel role in stimulating transcriptional elongation in vitro through interactions with the elongation factors P-TEFb and Tat-SF1; however, the mechanism remains unknown [1]. These factors are conserved in Saccharomyces cerevisiae, a fact that suggests that a similar interaction may occur in yeast to stimulate transcriptional elongation in vivo. To address this possibility we have looked for evidence of a role for the yeast Tat-SF1 homolog, Cus2, and the U2 snRNA in regulating transcription. Specifically, we have performed a genetic analysis to look for functional interactions between Cus2 or U2 snRNA and the P-TEFb yeast homologs, the Bur1/2 and Ctk1/2/3 complexes. In addition, we have analyzed Cus2-deleted or -overexpressing cells and U2 snRNA mutant cells to determine if they show transcription-related phenotypes similar to those displayed by the P-TEFb homolog mutants. In no case have we been able to observe phenotypes consistent with a role for either spliceosomal factor in transcription elongation. Furthermore, we did not find evidence for physical interactions between the yeast U2 snRNP factors and the P-TEFb homologs. These results suggest that in vivo, S. cerevisiae do not exhibit functional or physical interactions similar to those exhibited by their mammalian counterparts in vitro. The significance of the difference between our in vivo findings and the previously published in vitro results remains unclear; however, we discuss the potential importance of other factors, including viral proteins, in mediating the mammalian interactions

Charged tmRNA but not tmRNA-mediated proteolysis is essential for Neisseria gonorrhoeae viability

tmRNA, through its tRNA and mRNA properties, adds short peptide tags to abnormal proteins, targeting these proteins for proteolytic degradation. Although the conservation of tmRNA throughout the bacterial kingdom suggests that it must provide a strong selective advantage, it has not been shown to be essential for any bacterium. We report that tmRNA is essential in Neisseria gonorrhoeae. Although tagging per se appears to be required for gonococcal viability, tagging for proteolysis does not. This suggests that the essential roles of tmRNA in N.gonorrhoeae may include resolving stalled translation complexes and/or preventing depletion of free ribosomes. Although derivatives of N.gonorrhoeae expressing Escherichia coli tmRNA as their sole tmRNA were isolated, they appear to form colonies only after acquiring an extragenic suppressor(s)

Proximity of conserved U6 and U2 snRNA elements to the 5′ splice site region in activated spliceosomes

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA–RNA interactions, little is known about the tertiary structure of this RNA–RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5′ end of a pre-mRNA intron, to map RNA–RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step