Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing

Box C/D small nucleolar RNPs catalyse 2′-O-methylation of eukaryotic ribosomal RNA. A large-scale analysis of yeast box C/D snoRNAs reveals conserved ‘extra base-pairing' between snoRNAs and regions adjacent to their rRNA methylation site and points to a role for the non-catalytic protein subunits Nop56 and Nop58 in rRNA binding

Puf6 primes 60S pre-ribosome nuclear export at low temperature

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.ISSN:2041-172

A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease

The 5′-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5′-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5′-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A(3). Processing from A(3) requires the ‘A(3)-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A(3)-cluster factors were not crosslinked close to site A(3), but bound sites around the 5.8S 3′- and 25S 5′-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5′-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A(3)-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A(3) cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding

Identification of conserved secondary structures and expansion segments in enod40 RNAs reveals new enod40 homologues in plants

enod40 is a plant gene that participates in the regulation of symbiotic interaction between leguminous plants and bacteria or fungi. Furthermore, it has been suggested to play a general role in non-symbiotic plant development. Although enod40 seems to have multiple functions, being present in many land plants, the molecular mechanisms of its activity are unclear; they may be determined though, by short peptides and/or RNA structures encoded in the enod40 genes. We utilized conserved RNA structures in enod40 sequences to search nucleotide sequence databases and identified a number of new enod40 homologues in plant species that belong to known, but also, to yet unknown enod40-containing plant families. RNA secondary structure predictions and comparative sequence analysis of enod40 RNAs allowed us to determine the most conserved structural features, present in all known enod40 genes. Remarkably, the topology and evolution of one of the conserved structural domains are similar to those of the expansion segments found in structural RNAs such as rRNAs, RNase P and SRP RNAs. Surprisingly, the enod40 RNA structural elements are much more stronger conserved than the encoded peptides. This finding suggests that some general functions of enod40 gene could be determined by the encoded RNA structure, whereas short peptides may be responsible for more diverse functions found only in certain plant families

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of the cytoplasmic translation initiation factor eIF5b/Fun12. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associate with Fun12 and form 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP-hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together

The Nrd1-like protein Seb1 coordinates cotranscriptional 3′ end processing and polyadenylation site selection

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3 end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3 end processing factors, is enriched at the 3 end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3 untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3 end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation