In Advance

[ARTICLE] Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

Sibert, B. S., Fischel-Ghodsian, N., Patton, J. R. — 2008-07-22

Pseudouridine synthase 1 (Pus1p) is an enzyme that converts uridine to Pseudouridine () in tRNA and other RNAs in eukaryotes. The active site of Pus1p is composed of stretches of amino acids that are highly conserved and it is hypothesized that mutation of select residues would impair the enzyme's ability to catalyze the formation of . However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all synthases tested. To determine the requirements for particular amino acids at certain absolutely conserved positions in Pus1p, three residues (R116, Y173, R267) that correspond to amino acids known to compose the active site of TruA, a bacterial synthase that is homologous to Pus1p, were mutated in human Pus1p (hPus1p). The effects of those mutations were determined with three different in vitro assays of pseudouridylation and several tRNA substrates. Surprisingly, it was found that each of these components of the hPus1p active site could tolerate certain amino acid substitutions and in fact most mutants exhibited some activity. The most active mutants retained near wild-type activity at positions 27 or 28 in the substrate tRNA, but activity was greatly reduced or absent at other positions in tRNA readily modified by wild-type hPus1p.

[ARTICLE] The highest affinity binding site of small protein B on transfer messenger RNA is outside the tRNA domain

Metzinger, L., Hallier, M., Felden, B. — 2008-07-22

Eubacterial ribosomes stalled on defective mRNAs are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein B (SmpB) and transfer messenger RNA (tmRNA). A series of tmRNA variants with deletions in each structural domain were produced. Their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of SmpB. Dissociation constants between these RNAs and SmpB from Aquifex aeolicus were derived by surface plasmon resonance (SPR) combined with filter binding assays. Three independent experimental evidences, including filter binding assays, SPR, and concentration titrations of the RNA–protein reactivity changes toward structural probes, indicate that the binding site that has the highest affinity for the protein is located outside the tRNA domain, upstream of the internal tag. The minimal tmRNA fragment that contains this high affinity site for SmpB, and also contains another site of lower affinity, includes the tag reading frame and three downstream pseudoknots that form a ring structure in solution.

[ARTICLE] Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo

Ramirez, A., Shuman, S., Schwer, B. — 2008-07-22

Yeast and human Clp1 proteins are homologous components of the mRNA 3'-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5'-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5'-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1 strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5'-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.

[ARTICLE] A new regulatory circuit in ribosomal protein operons: S2-mediated control of the rpsB-tsf expression in vivo

2008-07-22

Autogenous regulation is a general strategy of balancing ribosomal protein synthesis in bacteria. Control mechanisms have been studied in detail for most of ribosomal protein operons, except for rpsB-tsf encoding essential r-protein S2 and elongation factor Ts, where even the promoter has remained unknown. By using single-copy translational fusions with the chromosomal lacZ gene and Western-blot analysis, we demonstrate here that S2 serves as a negative regulator of both rpsB and tsf expression in vivo, acting at a single target within the rpsB 5'-untranslated region (5'-UTR). As determined by primer extension, transcription of the Escherichia coli rpsB-tsf operon starts 162 nucleotides upstream of the rpsB initiation codon at a single promoter TGTGGTATAAA belonging to the extended –10 promoter class. Both the promoter signature and the 5'-UTR structure of the rpsB gene appear to be highly conserved in -proteobacteria. Deletion analysis of the rpsB 5'-UTR within rpsB'-'lacZ fusions has revealed that an operator region involved in the S2 autoregulation comprises conserved structural elements located upstream of the rpsB ribosome binding site. The S2-mediated autogenous control is impaired in rpsB mutants and, more surprisingly, in the rpsA mutant producing decreased amounts of truncated r-protein S1 (rpsA::IS10), indicating that S2 might act as a repressor in cooperation with S1.

[ARTICLE] Trans-splicing versatility of the Ll.LtrB group II intron

Belhocine, K., Mak, A. B., Cousineau, B. — 2008-07-22

Group II introns are found in organelles, bacteria, and archaea. Some harbor an open reading frame (ORF) with reverse transcriptase, maturase, and occasionally endonuclease activities. Group II introns require the assistance of either intron-encoded or free-standing maturases to excise from primary RNA transcripts in vivo. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA sites by retrohoming or retrotransposition. Group II introns are also hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs—U1, U2, U4, U5, and U6) that are part of the spliceosome. The ability of some fragmented group II introns to undergo splicing in trans supports the theory that the snRNAs evolved from portions of group II introns. Here, we developed a Tn5-based genetic screen to explore the trans-splicing potential of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. Proficient trans-splicing variants of Ll.LtrB were selected using a highly sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support splicing in trans, showing that this intron is remarkably more tolerant to fragmentation than expected from the fragmentation sites uncovered within natural trans-splicing group II introns. This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo.

[ARTICLE] On the mechanism of induction of heterochromatin by the RNA-binding protein vigilin

Zhou, J., Wang, Q., Chen, L.-L., Carmichael, G. G. — 2008-07-22

Vigilin is an RNA-binding protein localized to both the cytoplasm and the nucleus and has been previously implicated in heterochromatin formation and chromosome segregation. We demonstrate here that the C-terminal domain of human vigilin binds to the histone methyltransferase SUV39H1 in vivo. This association is independent of RNA and maps to a site on vigilin that is not involved in its interaction with several other known protein partners. Cells that express high levels of the C-terminal fragment display chromosome segregation defects, and ChIP analyses show changes in the status of pericentric β-satellite and rDNA chromatin from heterochromatic to more euchromatic form. Finally, a cell line with inducible expression of the vigilin C-terminal fragment displays inducible alterations in β-satellite chromatin. These and other results lead us to present a new model for vigilin-mediated, RNA-induced gene silencing.