One of the most important tasks of any cell is to synthesize ribosomes. In eukaryotes, this process occurs sequentially in the nucleolus, the nucleoplasm and the cytoplasm. It involves the transcription and processing of pre-ribosomal RNAs, their proper folding and assembly with ribosomal proteins and the transport of the resulting pre-ribosomal particles to the cytoplasm where final maturation events occur. In addition to the protein and RNA constituents of the mature cytoplasmic ribosomes, this intricate process requires the intervention of numerous protein and small RNA trans-acting factors. These transiently interact with pre-ribosomal particles at various stages of their maturation. Most of the constituents of pre-ribosomal particles have probably now been identified and research in the field is starting to unravel the timing of their intervention and their precise mode of action. Moreover, quality control mechanisms are being discovered that monitor ribosome synthesis and degrade the RNA components of defective pre-ribosomal particles.

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5′-3′ resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5′-3′ resection at the shortest telomeres.

The model for telomere shortening at each replication cycle is currently incomplete, and the exact contribution of the telomeric 3' overhang to the shortening rate remains unclear. Here, we demonstrate key steps of the mechanism of telomere replication in Saccharomyces cerevisiae. By following the dynamics of telomeres during replication at near-nucleotide resolution, we find that the leading-strand synthesis generates blunt-end intermediates before being 5'-resected and filled in. Importantly, the shortening rate is set by positioning the last Okazaki fragments at the very ends of the chromosome. Thus, telomeres shorten in direct proportion to the 3' overhang lengths of 5-10 nucleotides that are present in parental templates. Furthermore, the telomeric protein Cdc13 coordinates leading- and lagging-strand syntheses. Taken together, our data unravel a precise choreography of telomere replication elucidating the DNA end-replication problem and provide a framework to understand the control of the cell proliferation potential.

It is generally assumed that, in Saccharomyces cerevisiae, immature 40S ribosomal subunits are not competent for translation initiation. Here, we show by different approaches that, in wild-type conditions, a portion of pre-40S particles (pre-SSU) associate with translating ribosomal complexes. When cytoplasmic 20S pre-rRNA processing is impaired, as in Rio1p-or Nob1p-depleted cells, a large part of pre-SSUs is associated with translating ribosomes complexes. Loading of pre-40S particles onto mRNAs presumably uses the canonical pathway as translation-initiation factors interact with 20S pre-rRNA. However, translation initiation is not required for 40S ribosomal subunit maturation. We also provide evidence suggesting that cytoplasmic 20S pre-rRNAs that associate with translating complexes are turned over by the no go decay (NGD) pathway, a process known to degrade mRNAs on which ribosomes are stalled. We propose that the cytoplasmic fate of 20S pre-rRNA is determined by the balance between pre-SSU processing kinetics and sensing of ribosome-like particles loaded onto mRNAs by the NGD machinery, which acts as an ultimate ribosome quality check point.

Highlights d Antisense transcription leads to À1/+1 nucleosome sliding, reducing sense initiation d H3K36me3 nucleosomes are differently positioned compared to H3K18ac nucleosomes d RSC recruitment to À1/+1 nucleosomes is modulated by histone acetylation levels d 20% of S. cerevisiae genes are significantly repressed by the proposed mechanism

Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.

High noncoding readthrough transcription leads to ARS chromatin regulation Given the links between replication initiation and chromatin structure, we then analyzed the effects of the Nrd1 depletion-induced transcription readthrough into replication origins on nucleosome positioning. Chromatin was extracted from Nrd1-AA cells either untreated or treated for 1 h with rapamycin and digested Noncoding transcription and replication program Genome Research 1883

RNA polymerase II (RNAP II) non-coding transcription is now known to cover almost the entire eukaryotic genome, a phenomenon referred to as pervasive transcription. As a consequence, regions previously thought to be non-transcribed are subject to the passage of RNAP II and its associated proteins for histone modification. This is the case for the nucleosome-depleted regions (NDRs), which provide key sites of entry into the chromatin for proteins required for the initiation of coding gene transcription and DNA replication. In this review, recent data on the effects of pervasive transcription through NDRs are summarized and a model is proposed to explain how RNAP II-driven transcription is able to modify the nucleosomes flanking the NDRs, leading to nucleosome repositioning and NDR closure. Even though much of the mechanistic detail underlying these events remains to be elucidated, such a model provides a basis to explain how non-coding transcription through NDRs can regulate the initiation of coding gene expression and DNA replication.