Alain Jacquier

Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase

Since detection of an RNA molecule is the major criterion to define transcriptional activity, the fraction of the genome that is expressed is generally considered to parallel the complexity of the transcriptome. We show here that several supposedly silent intergenic regions in the genome of S. cerevisiae are actually transcribed by RNA polymerase II, suggesting that the expressed fraction of the genome is higher than anticipated. Surprisingly, however, RNAs originating from these regions are rapidly degraded by the combined action of the exosome and a new poly(A) polymerase activity that is defined by the Trf4 protein and one of two RNA binding proteins, Air1p or Air2p. We show that such a polyadenylation-assisted degradation mechanism is also responsible for the degradation of several Pol I and Pol III transcripts. Our data strongly support the existence of a posttranscriptional quality control mechanism limiting inappropriate expression of genetic information.

Widespread bidirectional promoters are the major source of cryptic transcripts in yeast

Pervasive and hidden transcription is widespread in eukaryotes, but its global level, the mechanisms from which it originates and its functional significance are unclear. Cryptic unstable transcripts (CUTs) were recently described as a principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae. These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1–exosome–TRAMP complexes. Although CUT degradation mechanisms have been analysed in detail, the genome-wide distribution at the nucleotide resolution and the prevalence of CUTs are unknown. Here we report the first high-resolution genomic map of CUTs in yeast, revealing a class of potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic promoters. An RNA fraction highly enriched in CUTs was analysed by a 3′ Long-SAGE (serial analysis of gene expression) approach adapted to deep sequencing. The resulting detailed genomic map of CUTs revealed that they derive from extremely widespread and very well defined transcription units and do not result from unspecific transcriptional noise. Moreover, the transcription of CUTs predominantly arises within nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 5′ end. Our study of glycolysis genes, as well as recent results from the literature, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels.

Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure

The complete sequences of nine Saccharomyces cerevisiae mitochondrial introns, six of which carry long open reading frames, have already been published. We have recently determined the sequence of an intron in the large ribosomal mitochondrial RNA of Kluyveromyces thermotolerans (Jacquier et al., in preparation), which we found to be closely related to its S. cerevisiae counterpart. This latter result prompted us to undertake a systematic search for possible homologous elements in the other, available sequences with the help of an original computer program. A previously unsuspected wealth of evolutionarily conserved sequences and secondary structures was thus uncovered. Seven at least of the available sequences may be folded up into elaborate secondary structure models, the cores of which are nearly identical. These models result in bringing together the exon-intron junctions into relatively close spatial proximity and looping out either all or most of the sequences in open reading frame, when present. These results and their possible implications with respect to the mechanism of splicing are discussed in the light of available genetic and biochemical data.

Human L1 element target-primed reverse transcription in vitro

L1 elements are ubiquitous human transposons that replicate via an RNA intermediate. We have reconstituted the initial stages of L1 element transposition in vitro. The reaction requires only the L1 ORF2 protein, L1 3′ RNA, a target DNA and appropriate buffer components. We detect branched molecules consisting of junctions between transposon 3′ end cDNA and the target DNA, resulting from priming at a nick in the target DNA. 5′ junctions of transposon cDNA and target DNA are also observed. The nicking and reverse transcription steps in the reaction can be uncoupled, as priming at pre‐existing nicks and even double‐strand breaks can occur. We find evidence for specific positioning of the L1 RNA with the ORF2 protein, probably mediated in part by the polyadenosine portion of L1 RNA. Polyguanosine, similar to a conserved region of the L1 3′ UTR, potently inhibits L1 endonuclease (L1 EN) activity. L1 EN activity is also repressed in the context of the full‐length ORF2 protein, but it and a second cryptic nuclease activity are released by ORF2p proteolysis. Additionally, heterologous RNA species such as Alu element RNA and L1 transcripts with 3′ extensions are substrates for the reaction.

The complex eukaryotic transcriptome: unexpected pervasive transcription and novel small RNAs

Over the past few years, techniques have been developed that have allowed the study of transcriptomes without bias from previous genome annotations, which has led to the discovery of a plethora of unexpected RNAs that have no obvious coding capacities. There are many different kinds of products that are generated by this pervasive transcription; this Review focuses on small non-coding RNAs (ncRNAs) that have been found to be associated with promoters in eukaryotes from animals to yeast. After comparing the different classes of such ncRNAs described in various studies, the Review discusses how the models proposed for their origins and their possible functions challenge previous views of the basic transcription process and its regulation.

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell‐wall‐associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII‐mediated inhibition of translation and degradation of the stable spa mRNA by the double‐strand‐specific endoribonuclease III (RNase III). The 3′ end domain of RNAIII, partially complementary to the 5′ part of spa mRNA, efficiently anneals to spa mRNA through an initial loop–loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast.

Recent experiments have shown that gene repression can be correlated with relocation of genes to heterochromatin-rich silent domains. Here, we investigate whether nuclear architecture and spatial positioning can contribute directly to the transcriptional activity of a genetic locus in Saccharomyces cerevisiae. By disassembling telomeric silent domains without altering the chromatin-mediated silencing machinery, we show that the transcriptional activity of silencer–reporter constructs depends on intranuclear position. This demonstrates that telomeric silent domains are actively involved in transcriptional silencing. Employing fluorescent in situ hybridization (FISH) in combination with genetic assays, we demonstrate that telomeres control the establishment of transcriptional states by reversible partitioning with the perinuclear silencing domains. Anchoring telomeres interferes with their ability to assume an active state, whereas disassembly of silencing domains prevents telomeres from assuming a repressed state. Our data support a model in which domains of enriched transcriptional regulators allow genes to determine transcriptional states by spatial positioning.

Nog2p, a putative GTPase associated with pre‐60S subunits and required for late 60S maturation steps

Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP‐binding site, which is essential in vivo. Kinetic and steady‐state measurements of the levels of pre‐rRNAs in Nog2p‐depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SBS and 7SS precursors. We found Nog2p physically associated with large pre‐60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP‐binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre‐60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP‐dependent association of Nog2p with the pre‐ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.

Sequential Protein Association with Nascent 60S Ribosomal Particles

ABSTRACT Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1

Small nucleolar RNAs (snoRNAs) are intron encoded or expressed from monocistronic independent transcription units, or, in the case of plants, from polycistronic clusters. We show that the snR190 and U14 snoRNAs from the yeast Saccharomyces cerevisiae are co‐transcribed as a dicistronic precursor which is processed by the RNA endonuclease Rnt1, the yeast ortholog of bacterial RNase III. RNT1 disruption results in a dramatic decrease in the levels of mature U14 and snR190 and in accumulation of dicistronic snR190–U14 RNAs. Addition of recombinant Rnt1 to yeast extracts made from RNT1 disruptants induces the chase of dicistronic RNAs into mature snoRNAs, showing that dicistronic RNAs correspond to functional precursors stalled in the processing pathway. Rnt1 cleaves a dicistronic transcript in vitro in the absence of other factors, separating snR190 from U14. Thus, one of the functions of eukaryotic RNase III is, as for the bacterial enzyme, to liberate monocistronic RNAs from polycistronic transcripts.