Biomedical subjects

Anthony K Henras

Publications and source records attributed to Anthony K Henras.

7 recordsLinked to original sources

PubMed2006

Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome.

The nuclear exosome is involved in a large number of RNA processing and surveillance pathways. RNase III cleavage intermediates destined to be 3'-processed or degraded can be detected when the Rrp6p subunit of the nuclear exosome is absent. Here we show that these processing and degradation intermediates are polyadenylated, and that their polyadenylation is dependent on the activity of Trf4p and Trf5p, two variant poly(A) polymerases. Polyadenylation of cleavage intermediates was inhibited when Trf4p was absent, and reduced to various extents in the absence of Trf5p, suggesting that these two poly(A) polymerases play functionally distinct roles in the polyadenylation of these RNA species. Finally, in the absence of Trf4p, we observed 3'-extended forms of the U4 snRNA that are similar to those observed in the absence of Rrp6p. These results suggest that polyadenylation of RNA processing intermediates plays a functional role in RNA processing pathways and is not limited to RNA surveillance functions.

Animals↗

PubMed2005

Multiple RNA surveillance pathways limit aberrant expression of iron uptake mRNAs and prevent iron toxicity in S. cerevisiae.

Tight regulation of the expression of mRNAs encoding iron uptake proteins is essential to control iron homeostasis and avoid intracellular iron toxicity. We show that many mRNAs encoding iron uptake or iron mobilization proteins are expressed in iron-replete conditions in the absence of the S. cerevisiae RNase III ortholog Rnt1p or of the nuclear exosome component Rrp6p. Extended forms of these mRNAs accumulate in the absence of Rnt1p or of the 5'-->3' exonucleases Xrn1p and Rat1p, showing that multiple degradative pathways contribute to the surveillance of aberrant forms of these transcripts. RNase III-deficient cells are hypersensitive to high iron concentrations, suggesting that Rnt1p-mediated RNA surveillance is required to prevent iron toxicity. These results show that RNA surveillance through multiple ribonucleolytic pathways plays a role in iron homeostasis in yeast to avoid the potentially toxic effects of the expression of the iron starvation response in iron-replete conditions.

Drug-Related Side Effects and Adverse Reactions↗

PubMed2005

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III.

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.

Base Sequence↗

PubMed2005

A conserved major groove antideterminant for Saccharomyces cerevisiae RNase III recognition.

Rnt1p, the only known Saccharomyces cerevisiae RNase III double-stranded RNA endonuclease, plays important roles in the processing of precursors of ribosomal RNAs and small nuclear and nucleolar RNAs and in the surveillance of unspliced pre-mRNAs. Specificity of cleavage by Rnt1p relies on the presence of RNA tetraloop structures with the consensus sequence AGNN at the top of the target dsRNA. The sequences of 79 fungal RNase III substrates were inspected to identify additional conserved sequence elements or antideterminants that may contribute to Rnt1p recognition of the double-stranded RNA. Surprisingly, U-A sequences at the base pair adjacent to the conserved terminal tetraloop (closing base pair) were found to be absent from all but one inspected sequence. Analysis of chemically modified variants of the closing base pair showed that the presence of exocyclic groups in the major groove of purines 3' to the last nucleotide of the tetraloop inhibits Rnt1p cleavage without strongly inhibiting Rnt1p binding. We propose that these groups interfere with the recognition of the RNA substrate by the catalytic domain of Rnt1p. These results identify exocyclic groups of purines in the major groove downstream of the tetraloop as a major antideterminant in S. cerevisiae RNase III activity, and suggest a rationale for their apparent counter selection in RNA processing sites.

Adenine↗

PubMed2004

Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs.

Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited. In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p. We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other. We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.

Blotting, Western↗

PubMed2004

A cotranscriptional model for 3'-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor.

Cleavage of the Saccharomyces cerevisiae primary ribosomal RNA (rRNA) transcript in the 3' external transcribed spacer (ETS) by Rnt1p generates the 35S pre-rRNA, the earliest detectable species in the pre-rRNA processing pathway. In this study we show that Rnt1p is concentrated in a subnucleolar dot-shaped territory distinct from the nucleolar body. The 35S pre-rRNA is localized at the periphery of the Rnt1p dot, in a pattern that suggests a diffusion of the 35S pre-rRNA from the site of Rnt1p processing. When plasmid-borne versions of the rDNA are used to express rRNAs, the Rnt1p territory reorganizes around these plasmids, suggesting a close association between Rnt1p and the plasmid-borne rDNA units. Rnt1p was found associated with the endogenous rDNA by chromatin immunoprecipitation. Deletion of functionally important Rnt1p domains result in a loss of the dot-shaped territory, showing that this subnucleolar territory corresponds to a functional site of processing. These results show that a large fraction of Rnt1p is localized at the site of transcription of the rDNA, suggesting that the cleavage of the primary pre-rRNA transcript to generate the 35S pre-rRNA is a cotranscriptional event.

Cell Nucleolus↗

PubMed2004

RNA structure and function in C/D and H/ACA s(no)RNPs.

From archaea to humans, C/D- and H/ACA-type small ribonucleoprotein particles play key roles in crucial RNA processing events. Various such particles are required for pre-rRNA cleavage steps and/or for chemical modification of rRNAs, spliceosomal small nuclear RNAs, tRNAs and perhaps even mRNAs. Each C/D-type particle contains a small RNA possessing conserved C and D, as well as related C' and D', sequence motifs, whereas each H/ACA-type particle contains a small RNA featuring conserved H and ACA sequence elements. Recently published studies highlight the importance of sequence and structural elements of these RNAs in the localization, activity and assembly of the ribonucleoprotein particles. A novel sequence element, the Cajal body box, found at the apex of stem structures within a subset of H/ACA small RNAs, mediates the specific retention of particles containing these elements inside nucleoplasmic Cajal bodies. Two highly conserved elements, the m1 and m2 boxes, have been identified in the 3' stem of the atypical H/ACA snR30/U17 RNAs. These conserved sequence elements are necessary for early pre-rRNA cleavage events and consequently for mature 18S rRNA production. Finally, convincing evidence has been provided that the conserved C and D sequence motifs of C/D-type small RNAs fold into a helix-bulge-helix structure, called a kink-turn, that provides a platform for assembly of C/D-type ribonucleoprotein particles.

Animals↗