rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events

A bona fide La protein is required for embryogenesis in Arabidopsis thaliana

Searches in the Arabidopsis thaliana genome using the La motif as query revealed the presence of eight La or La-like proteins. Using structural and phylogenetic criteria, we identified two putative genuine La proteins (At32 and At79) and showed that both are expressed throughout plant development but at different levels and under different regulatory conditions. At32, but not At79, restores Saccharomyces cerevisiae La nuclear functions in non-coding RNAs biogenesis and is able to bind to plant 3′-UUU-OH RNAs. We conclude that these La nuclear functions are conserved in Arabidopsis and supported by At32, which we renamed as AtLa1. Consistently, AtLa1 is predominantly localized to the plant nucleoplasm and was also detected in the nucleolar cavity. The inactivation of AtLa1 in Arabidopsis leads to an embryonic-lethal phenotype with deficient embryos arrested at early globular stage of development. In addition, mutant embryonic cells display a nucleolar hypertrophy suggesting that AtLa1 is required for normal ribosome biogenesis. The identification of two distantly related proteins with all structural characteristics of genuine La proteins suggests that these factors evolved to a certain level of specialization in plants. This unprecedented situation provides a unique opportunity to dissect the very different aspects of this crucial cellular activity

Poly A- Transcripts Expressed in HeLa Cells

BACKGROUND: Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3' poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3' poly A tail; 3) applying the 454 sequencing technology for massive 3' EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3' ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3' poly A tail. Most of the poly A- 3' ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3' ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. CONCLUSION/SIGNIFICANCE: Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts

Non-coding RNA annotation of the genome of Trichoplax adhaerens

A detailed annotation of non-protein coding RNAs is typically missing in initial releases of newly sequenced genomes. Here we report on a comprehensive ncRNA annotation of the genome of Trichoplax adhaerens, the presumably most basal metazoan whose genome has been published to-date. Since blast identified only a small fraction of the best-conserved ncRNAs—in particular rRNAs, tRNAs and some snRNAs—we developed a semi-global dynamic programming tool, GotohScan, to increase the sensitivity of the homology search. It successfully identified the full complement of major and minor spliceosomal snRNAs, the genes for RNase P and MRP RNAs, the SRP RNA, as well as several small nucleolar RNAs. We did not find any microRNA candidates homologous to known eumetazoan sequences. Interestingly, most ncRNAs, including the pol-III transcripts, appear as single-copy genes or with very small copy numbers in the Trichoplax genome