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Summary Spermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3′ untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways., Graphical abstract, Highlights • NANOS2 is a 3′UTR mRNA binding protein and recognizes the AUKAAWU consensus • CCR4-NOT deadenylase complex is the primary interactor of NANOS2 in spermatogonia • NANOS2 binding results in mRNA destabilization • NANOS2 regulates key spermatogonial signaling and metabolic pathways, Biological sciences; Molecular biology; Developmental biology; Omics; Transcriptomics

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the "neck" and "beak" regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli, translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ΔybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production.

Summary Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5′ ends of mRNAs. Following glucose withdrawal, 5′ binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5′ RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress., Graphical Abstract, Highlights • A quantitative proteomic approach reveals global stress-induced changes in RNA binding • Translation shutdown is driven by rapid loss of mRNA binding by key initiation factors • Heat shock induces general mRNA degradation facilitated by Xrn1, All organisms live in continuously changing environments, and RNA-binding proteins are critically important in responding to resulting stresses. Bresson et al. followed both global and specific changes in RNA-protein interactions over short times following glucose starvation or heat shock. The results reveal mechanisms underlying translational control of gene expression during stress.

Summary Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5′ enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding., Graphical Abstract, Highlights • Structures in the nascent RNA correlate with rapid elongation by RNAPI in vivo • Stable RNA structures limit RNAPI backtracking in vitro • GC content in the transcription bubble tunes transcription elongation rate • Nascent transcript folding modulates dynamics of all three RNAPs in vivo, Transcription elongation is a stochastic process that can go backward as well as forward. Here Turowski et al. report that folding of the nascent transcript immediately behind the polymerase resists backtracking and promotes forward movement. This results in faster elongation relative to unstructured sequences and is potentially important for cotranscriptional processing.

During transcription, the nascent transcript behind an elongating RNA polymerase (RNAP) can invade the DNA duplex and hybridize with the complementary DNA template strand, generating a three-stranded "R-loop" structure, composed of an RNA:DNA duplex and an unpaired non-template DNA strand. R-loops can be strongly associated with actively transcribed loci by all RNAPs including the mitochondrial RNA polymerase (mtRNAP). In this chapter, we describe two protocols for the detection of RNA:DNA hybrids in living budding yeast cells, one that uses conventional chromatin immunoprecipitation (ChIP-qPCR) and one that uses DNA:RNA immunoprecipitation (DRIP-qPCR). Both protocols make use of the S9.6 antibody, which is believed to recognize the intermediate A/B helical RNA:DNA duplex conformation, with no sequence specificity.

Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo. High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

Eukaryotic ribosomes are synthesized in a complex, multistep pathway. This begins with transcription of the rDNA genes by a specialized RNA polymerase, accompanied by the cotranscriptional binding of large numbers of ribosome synthesis factors, small nucleolar RNAs and ribosomal proteins. Cleavage of the nascent transcript releases the early pre-40S and pre-60S particles, which acquire export competence in the nucleoplasm prior to translocation through the nuclear pore complexes and final maturation to functional ribosomal subunits in the cytoplasm. This review will focus on the many and complex interactions occurring during pre-rRNA synthesis, particularly in budding yeast in which the pathway is best understood. WIREs RNA 2015, 6:129–139. doi: 10.1002/wrna.1263 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as ‘new system’ names.

In this issue of Molecular Cell, Webster et al. (2018) and Yi et al. (2018) dissect the mechanisms underlying cytoplasmic mRNA deadenylation by the Ccr4-Not (CNOT) complex. Crucial to this process is the poly(A) binding protein Pab1/PABPC1, which both stimulates and suppresses the activity of different deadenylases.

Highlights • A wide range of in vivo targets for the exosome complex has been established. • RNA polymerase III transcripts have emerged as major substrates. • The human nucleus has spatially localized forms of the exosome, with matching cofactors. • Structural analyses reveal a highly conserved RNA path through the eukaryotic exosome., In eukaryotes, the exosome complex degrades RNA backbones and plays key roles in RNA processing and surveillance. It was predicted that RNA substrates are threaded through a central channel. This pathway is conserved between eukaryotic and archaeal complexes, even though nuclease activity was lost from the nine-subunit eukaryotic core (EXO-9) and transferred to associated proteins. The exosome cooperates with nuclear and cytoplasmic cofactors, including RNA helicases Mtr4 and Ski2, respectively. Structures of an RNA-bound exosome and both helicases revealed how substrates are channeled through EXO-9 to the associated nuclease Rrp44. Recent high-throughput analyses provided fresh insights relating exosome structure to its diverse in vivo functions. They also revealed surprisingly high degradation rates for newly synthesized RNAs, particularly RNA polymerase III transcripts.

Pre-ribosomal particles evolve in the nucleus through transient interaction with biogenesis factors, before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle purified from Saccharomyces cerevisiae through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-electron microscopy reconstruction of the Arx1-particle at 11.9 Å resolution reveals regions of extra densities on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immuno-electron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation.

Summary The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. Here we apply in vivo RNA crosslinking (CRAC) to the nucleases (Rrp44, Rrp6), two structural subunits (Rrp41, Csl4) and a cofactor (Trf4) of the yeast exosome. Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of non-coding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome indicating that substrates follow multiple pathways to the nucleases., Graphical Abstract Highlights ► The in vivo target range was identified for the exosome nuclease complex ► The endonuclease and exonuclease activities of Rrp44/Dis3 function cooperatively ► Comparison of Rrp6 and core exosome suggests multiple substrate recruitment pathways ► Pre-tRNA and other RNAs transcribed by Pol III emerge as major exosome targets

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3’ end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation. Tiling arrays and RNAPII mapping data revealed 3’ extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression., Author Summary Npl3 is a yeast mRNA binding protein with many reported functions in RNA processing. We wanted to identify direct targets and therefore combined analyses of the transcriptome-wide effects of the loss of Npl3 on gene expression with UV crosslinking and bioinformatics to identify RNA-binding sites for Npl3. We found that Npl3 binds diverse sites on large numbers of transcripts, and that the loss of Npl3 results in transcriptional readthrough on many genes. One effect of this transcription readthrough is that the expression of numerous flanking genes is strongly down regulated. This underlines the importance of faithful termination for the correct regulation of gene expression. The effects of the loss of Npl3 are seen on both mRNAs and non-protein coding RNAs. These have distinct but overlapping termination mechanisms, with both classes requiring Npl3 for correct RNA packaging.

Individual microRNAs (miRNAs) are rapidly down-regulated during conditions of cellular activation and infection, but factors mediating miRNA turnover are poorly understood. Infection of mouse cells with murine cytomegalovirus (MCMV) induces the rapid down-regulation of an antiviral cellular miRNA, miR-27. Here, we identify a transcript produced by MCMV that binds to miR-27 and mediates its degradation. UV-crosslinking and high-throughput sequencing [CRAC (UV-crosslinking and analysis of cDNA)] identified MCMV RNA segments associated with the miRNA-binding protein Argonaute 2 (Ago2). A cluster of hits mapped to a predicted miR-27-binding site in the 3′UTR of the previously uncharacterized ORF, m169. The expression kinetics of the m169 transcript correlated with degradation of miR-27 during infection, and m169 expression inhibited miR-27 functional activity in a reporter assay. siRNA knockdown of m169 demonstrated its requirement for miR-27 degradation following infection and did not affect other host miRNAs. Substitution of the miR-27-binding site in m169 to create complementarity to a different cellular miRNA, miR-24, resulted in down-regulation of only miR-24 following infection. The m169 transcript is cytoplasmic, capped, polyadenylated, and interacts with miRNA-27 through seed pairing: characteristic features of the normal messenger RNA (mRNA) targets of miRNAs. This virus–host interaction reveals a mode of miRNA regulation in which a mRNA directs the degradation of a miRNA. We speculate that RNA-mediated miRNA degradation could be a more general viral strategy for manipulating host cells.

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 A3. Processing from A3 requires the ‘A3-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A3-cluster factors were not crosslinked close to site A3, 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 A3-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A3 cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.

2′-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C′/D′ motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This ‘extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA–protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C′/D′ motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA–rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.

To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.

The nuclear RNA polymerase II surveillance system targets polymerase III transcripts The exosome and Trf4/5–Air1/2–Mtr4 polyadenylation (TRAMP) complexes together with the Nrd1–Nab3 RNA-binding heterodimer have an important role in RNA surveillance. Here, the global analysis of Nrd1, Nab3 and Trf4 binding sites identifies targets for the nuclear surveillance system, including mRNAs, ncRNAs and RNA polymerase III transcripts., A key question in nuclear RNA surveillance is how target RNAs are recognized. To address this, we identified in vivo binding sites for nuclear RNA surveillance factors, Nrd1, Nab3 and the Trf4/5–Air1/2–Mtr4 polyadenylation (TRAMP) complex poly(A) polymerase Trf4, by UV crosslinking. Hit clusters were reproducibly found over known binding sites on small nucleolar RNAs (snoRNAs), pre-mRNAs and cryptic, unstable non-protein-coding RNAs (ncRNAs) (‘CUTs'), along with ∼642 predicted long anti-sense ncRNAs (asRNAs), ∼178 intergenic ncRNAs and, surprisingly, ∼1384 mRNAs. Five putative asRNAs tested were confirmed to exist and were stabilized by loss of Nrd1, Nab3 or Trf4. Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites. Nrd1 and Nab3 were believed to be Pol II specific but, unexpectedly, bound many oligoadenylated Pol III transcripts, predominately pre-tRNAs. Depletion of Nrd1 or Nab3 stabilized tested Pol III transcripts and their oligoadenylation was dependent on Nrd1–Nab3 and TRAMP. Surveillance targets were enriched for non-encoded A-rich tails. These were generally very short (1–5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.

Summary The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3′ UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA., Graphical Abstract, Highlights • HuD enhances the efficiency of translation in a mTORC1-independent fashion • The small non-coding RNA Y3 is the predominant HuD target in motor neuron-like cells • Y3 sequesters HuD from polysomes and counteracts its translation enhancer activity • Y3 blocks the function of HuD in neuronal differentiation, Tebaldi et al. report that the neuronal RNA binding protein HuD increases translation. The small non-coding RNA Y3 is a molecular sponge of HuD, limiting its recruitment to polysomes and neuron differentiation. These findings uncover an alternative route to the mTORC1 pathway for translational control, tunable by a non-coding RNA.

Summary Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 3′ end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 5′ end maturation. Here, we identify Rrp17p as a previously unidentified 5′–3′ exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 3′ end formation. Rrp17p is required for efficient exonuclease digestion of the mature 5′ ends of 5.8SS and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.

Many RNA nucleases and helicases participate in ribosome biogenesis, but how they cooperate with each other is largely unknown. Here we report that in vivo cleavage of the yeast pre-rRNA at site D, the 3'-end of the 18 S rRNA, requires functional interactions between PIN (PilT N terminus) domain protein Nob1 and the DEAH box RNA helicase Prp43. Nob1 showed specific cleavage on a D-site substrate analogue in vitro, which was abolished by mutations in the Nob1 PIN domain or the RNA substrate. Genetic analyses linked Nob1 to the late pre-40 S-associated factor Ltv1, the RNA helicase Prp43, and its cofactor Pfa1. In strains lacking Ltv1, mutation of Prp43 or Pfa1 led to a striking accumulation of 20 S pre-rRNA in the cytoplasm due to inhibition of site D cleavage. This phenotype was suppressed by increased dosage of wild-type Nob1 but not by Nob1 variants mutated in the catalytic site. In ltv1/pfa1 mutants the 20 S pre-rRNA was susceptible to 3' to 5' degradation by the cytoplasmic exosome. This degraded into the 3' region of the 18 S rRNA, strongly indicating that the preribosomes are structurally defective.

From the earliest comparisons of RNA production with steady-state levels, it has been clear that cells transcribe more RNA than they accumulate, implying the existence of active RNA degradation systems. In general, RNA is degraded at the end of its useful life, which is long for a ribosomal RNA but very short for excised introns or spacer fragments, and is closely regulated for most mRNA species. RNA molecules with defects in processing, folding, or assembly with proteins are identified and rapidly degraded by the surveillance machinery. Because RNA degradation is ubiquitous in all cells, it is clear that it must be carefully controlled to accurately recognize target RNAs. How this is achieved is perhaps the most pressing question in the field.

Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3 exonuclease activity. We report that expression only of Rrp44 lacking 3-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endoexo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.

The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar availability. Unexpectedly, under repressive conditions the 3' region of the GAL10 coding sequence is trimethylated by Set1 on histone H3 K4, normally characteristic of 5' regions of actively transcribed genes. This reflects transcription of a long noncoding RNA (GAL10-ncRNA) that is reciprocal to GAL1 and GAL10 mRNAs and driven by the DNA-binding protein Reb1. Point mutations in predicted Reb1-binding sites abolished Reb1 binding and ncRNA synthesis. The GAL10-ncRNA is transcribed approximately once every 50 min and targeted for degradation by the TRAMP and exosome complexes, resulting in low steady-state levels (approximately one molecule per 14 cells). GAL10-ncRNA transcription recruits the methyltransferase Set2 and histone deacetylation activities in cis, leading to stable changes in chromatin structure. These chromatin modifications act principally through the Rpd3S complex to aid glucose repression of GAL1-10 at physiologically relevant sugar concentrations.

In yeast, three small nucleolar RNAs (snoRNAs) are essential for the processing of pre-ribosomal RNA—U3, U14 and snR30—whereas 72 non-essential snoRNAs direct site-specific modification of pre-rRNA. We applied a quantitative screen for alterations in the pre-ribosome association to all 75 yeast snoRNAs in strains depleted of eight putative helicases implicated in 40S subunit synthesis. For the modification-guide snoRNAs, we found no clear evidence for the involvement of these helicases in the association or dissociation of pre-ribosomes. However, the DEAD box helicase Rok1 was required specifically for the release of snR30. Point mutations in motif I, but not in motif III, of the helicase domain of Rok1 impaired the release of snR30, but this was less marked than in strains depleted of Rok1, and resulted in a dominant-negative growth phenotype. Dissociation of U3 and U14 from pre-ribosomes is also dependent on helicases, suggesting that release of the essential snoRNAs might differ mechanistically from release of the modification-guide snoRNAs. Keywords: ribosome biogenesis; RNA helicase; snoRNA

Microarray analyses of mRNAs over-expressed in strains lacking the nuclear exosome component Rrp6 identified the transcript encoding the ARE-binding protein Cth2, which functions in cytoplasmic mRNA stability. Subsequent northern analyses revealed that exosome mutants accumulate a 3'-extended transcript at the expense of the mature CTH2 mRNA. The 3' ends of the CTH2 mRNA were mapped to a [GU(3-5)](5) repeat, unlike any previously characterized polyadenylation site. CTH2 mRNA accumulation was not inhibited by mutations in 3'-cleavage and polyadenylation factors, Rna14, Rna15 and Pap1, which block accumulation of other mRNAs. The 3'-extended CTH2 pre-mRNA strongly accumulated in strains with mutations in the TRAMP4 polyadenylation complex or the Nrd1/Nab3/Sen1 complex, and contains multiple Nrd1 and Nab3 binding sites. CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5'-exonuclease Rat1. We propose that CTH2 mRNA is processed from a 3'-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/ Sen1 complexes. This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination.

The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNA(i)(Met) lacking a single m(1)A(58) modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNA(i)(Met), showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNA(i)(Met) by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein.

The discoveries that have had the greatest effect on my work over the past 20 years relate to the nuclear RNA surveillance systems, particularly the exosome complex and its cofactors in budding yeast. These insights were made possible, in large part, by technical progress; in the fields of proteomics, genetics, structural biology, high-throughput sequencing, and RNA-protein crosslinking—and this is typical of many recent advances in RNA biology. The exosome story started from genetic and biochemical analyses of the yeast ribosome synthesis pathway. Prior to 1994, a small number of ribosome synthesis factors had been identified, but detailed functions were known for very few of these, and none of the processing enzymes had been characterized. In 1994, our genetic and biochemical analyses revealed that the endonuclease RNase MRP acts to provide an entry site for the 5′ exonuclease Rat1 (Xrn2), which generates the 5′ end of the 5.8S rRNA. Following on from this work, a genetic screen performed in 1995 identified a mutant rrp4.1 (rRNA processing) that was defective in generating the 3′ end of the 5.8S rRNA. The heterogeneous nature of the intermediates seen in the rrp4.1 strain suggested that, similar to 5′ processing, the 3′ end of 5.8S was generated by a 3′ exonuclease acting from a downstream endonuclease cleavage site. Protein A tags had recently been developed and tagged Rrp4 was shown by Phil Mitchell to co-purify with a 3′ exonuclease, supporting this model. Subsequent work revealed that Rrp4 was the first identified component on the large RNA degradation complex—termed the exosome. Around 1996 several groups were working to develop mass-spectrometry (MS) for protein identification. This key technical advance, allowed the group of Matthias Mann at EMBL to identify five other exosome proteins associated with Rrp4; these were designated as Rrp41, 42, 43, and 44 (also known as Dis3), as well as Mtr3 (mRNA transport) which had been identified by the Tartakof lab via a mutant showing nuclear poly(A) accumulation. With continued improvements in MS technology, further components of the exosome had been identified by 1999; Rrp40, Rrp45, Rrp46, and Csl4. In addition, specific exosome-associated proteins were identified in the nuclear and cytoplasmic complexes, Rrp6 and Ski7, respectively, which had initially been identified by the Butler and Wickner labs. Together this indicated that a “core exosome” consisting of ten proteins (Rrp4, Rrp40–46, Mtr3, Csl4) was present in both the nucleus and cytoplasm, with Rrp6 and Ski7 specifically associated with the nuclear and cytoplasmic complexes, respectively. Many exosome subunits appeared to be related to other known nucleases. Biochemical and sequence analyses identified Rrp44 and Rrp6 as hydrolytic 3′ exonucleases, that are homologous to E. coli RNase R and RNase D, respectively. Subsequently it emerged that Rrp44 also has an additional endonuclease cleavage activity. Six of the exosome subunits were related in sequence to the phosphorolytic 3′ exonuclease RNase PH, but it currently appears that none of these are active in the yeast or human exosome. However, the best conserved RNase PH homolog Rrp41 retained its phosphorolytic nuclease activity in Archaea and plants. Biochemical analyses indicated that the exosome had only limited in vitro RNase activity, suggesting that its apparently strong in vivo activity might depend on the presence of activating cofactors. In the late 1990s, the Parker, Johnson, and Takada labs characterized the SKI complex, comprising the DEAH box RNA helicase Ski2 together with the tetratricopeptide repeat (TPR) protein Ski3 and the WD40 repeat protein Ski8. This complex acts together with Ski7 as a cofactor for the cytoplasmic exosome in mRNA turnover and surveillance. Yeast Ski7 shows homology to GTPases that function in translation, suggesting functionally important interaction with the ribosome, but is not clearly conserved to humans. Around the same time Mtr4, an RNA helicase related to Ski2 was identified as a nuclear exosome cofactor, by the Linder group and ourselves. The identification of these closely related nuclear and cytoplasmic helicases provided a satisfying symmetry. Unexpectedly, however, analyses in the mid 2000s by the Keller, Jacquier, and Anderson groups and ourselves, showed that Mtr4 is not present in a complex similar to SKI. Rather it forms the TRAMP (Trf, Air, Mtr4) polyadenylation complexes, together with a small Zn-finger protein, Air1 or Air2, and a poly(A) polymerase, Trf4 or Trf5, which add short oligo(A) tails to target RNAs. This activity presumably provides a single-stranded “landing pad” that helps initiate RNA degradation by the exosome—as previously shown for E. coli poly(A) polymerase. Analyses, by the Jansen and Vanacova groups among others, indicate that nuclear degradation in human cells is even more complicated, since Mtr4 associates with several small RNA binding proteins to generate further exosome cofactor complexes, in addition to TRAMP. The initial substrates for the exosome were all identified individually by northern hybridization in strains with genetic defects in exosome components or cofactors. These analyses revealed roles in 3′ processing of many stable RNAs, functions in nuclear and cytoplasmic RNA turnover, and quality control functions for stable RNA and mRNA precursors. Subsequent analyses, starting in the mid 2000's, made use of tiling microarrays, and then next generation sequencing, to examine transcriptome-wide phenotypes. This work, initially by the Steinmetz, Jansen, Ares, and Jacquier labs, greatly expanded the range of known substrates, identifying very large numbers of short-lived, long noncoding RNAs (lncRNAs) as targets for the TRAMP/exosome system. These can be generated from independent transcription units, or associated with the promoter and enhancer regions of other genes. The TRAMP/exosome surveillance system has therefore emerged as the guardian of the transcriptome, constantly clearing many of the thousands of lncRNAs that are generated in all eukaryotes tested to date. In addition, our transcriptome-wide RNA-protein crosslinking, and analyses of exosome mutants by the Libri lab both indicate high levels of degradation of precursors to stable RNAs, particularly pre-tRNAs. These appear to have surprisingly high rates of rejection by the nuclear quality control systems. In recent years, structural analyses have transformed our understanding of the exosome and its cofactors. Van Hoof and Parker first proposed that the exosome might resemble the proteosome in having a barrel structure with a central substrate channel and internal degradative active sites. Crystallography, particularly by the Conti and Lima groups, has demonstrated that the exosome core is indeed a barrel through which many RNA substrates pass. In the archaeal exosome, and probably also the plant complex, an internal exonuclease active site lies in Rrp41. Surprisingly, as first reported by the Seraphin lab in 2007, this seems not to be the case for the yeast and human exosomes, which appear to have acquired inactivating point mutations. Despite this, the geometry of the yeast RNA channel, and the pathway followed by the substrate RNA, is notably similar to the archaeal complex; even including the location of the (former) active site of the Rrp41 component. The exosome core might appear to simply be a tube to conduct RNA from the cofactors to the exonuclease active site of Rrp44 that is localized at the end of the ∼31 nt long central channel. However, the high structural conservation strongly suggests that there is more to discover about the functional interactions between the exosome and its many substrate RNAs. Recent structural analyses have been reported from the Conti group for the nuclear TRAMP and cytoplasmic Ski complexes. These indicate elegant arrangements in which the RNA substrate passes through a loop structure in the helicase in the complex (Mtr4 or Ski2, respectively), before being channeled into the lumen of the exosome. These helicase presumably help remove secondary structure and bound proteins from substrate RNAs. This is particularly important because only single stranded RNA molecules can enter the lumen of the exosome.

We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1-2 mutation, pre-60S particles were rapidly degraded following transfer to 37 degrees C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1-2 strains at 37 degrees C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1-2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.

We report that Ypl146cp/Nop53p is associated with pre-60S ribosomal complexes and localized to the nucleolus and nucleoplasm. In cells depleted of Nop53p synthesis of the rRNA components of the 60S ribosomal subunit is severely inhibited, with strikingly strong accumulation of the 7S pre-rRNA and a 5′ extended form of the 25S rRNA. In cells depleted of Nop53p pre-60S subunits accumulate in the nucleus. However, a heterokaryon assay demonstrated that Nop53p is not transferred between nuclei, indicating that it is not released into the cytoplasm. We conclude that Nop53p is a late-acting factor in the nuclear maturation of 60S ribosomal subunits, which is required for normal acquisition of export competence. The strong accumulation of preribosomes in the Nop53p-depleted strain further suggests that it may participate in targeting aberrant preribosomes to surveillance and degradation pathways.

Eukaryotic cells contain many different RNA species. Nuclear pre-mRNAs and cytoplasmic mRNAs carry genomic information to the protein synthesis machinery, whereas many stable RNA species have important functional roles. The mature, functional forms of these RNA species are generated by post-transcriptional processing, and evidence has been accumulating that there are functional links between the various processing pathways. This indicates that there are regulatory networks that coordinate different stages of RNA metabolism. This article describes the aims and results, to date, of the European RNOMICS project as an example of an integrated approach to investigate these links.

In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron–sulfur (Fe–S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe–S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe–S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe–S proteins.

Nob1p (Yor056c) is essential for processing of the 20S pre-rRNA to the mature 18S rRNA. It is part of a pre-40S ribosomal particle that is transported to the cytoplasm and subsequently cleaved at the 3′ end of mature 18S rRNA (D-site). Nob1p is also reported to participate in proteasome biogenesis, and it was therefore unclear whether its primary activity is in ribosome synthesis. In this work, we describe a homology model of the PIN domain of Nob1p, which structurally mimics Mg2+-dependent exonucleases despite negligible similarity in primary sequence. Insights gained from this model were used to design a point mutation that was predicted to abolish the postulated enzymatic activity. Cells expressing Nob1p with this mutation failed to cleave the 20S pre-rRNA. This supports both the significance of the structural model and the idea that Nob1p is the long-sought D-site endonuclease.

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.

The Noc1–4p proteins were previously reported to be involved in intranuclear and nucleocytoplasmic transport of pre-ribosomes. Using fold recognition and structural modeling, we show that Noc1–4p are largely comprised of α-helical repeats similar to HEAT repeats. Because other HEAT-repeat proteins play key roles in transport processes, this finding provides a plausible mechanistic explanation for the function of the Noc proteins.

Rea1 is an ATPase related to dynein motor proteins that has been implicated in the biogenesis of the 60S ribosomal subunit. A new cryo-EM study vividly demonstrates the power of structural methods, deciphering the role of Rea1 in monitoring key pre-60S maturation steps before the acquisition of export competence in budding yeast.

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.

Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.

Depletion of any of the essential Lsm proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5' and 3' exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5' and 3' degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly.

Strains carrying rna14.1 and rna15.2 mutations are defective in pre-mRNA 3' cleavage, polyadenylation, and transcription termination. Long extended read-through transcripts generated in rna14.1 and rna15.2 strains are greatly stabilized by depletion of Rrp41p, a core component of the exosome complex or the RNA helicase Dob1p/Mtr4p. The absence of the nuclear-specific exosome component, Rrp6p, from the rna14.1 strain gave a very different phenotype. Short polyadenylated pre-mRNAs were strongly stabilized, and these were functional for translation. Production of these mRNAs was suppressed by depletion of Rrp41p, indicating that they are the products of exosome processing followed by uncoupled polyadenylation. The balance between complete degradation of 3'-unprocessed pre-mRNAs and their processing to functional mRNAs is regulated, with degradation favored on glucose media.

The Pescadillo protein was identified via a developmental defect and implicated in cell cycle progression. Here we report that human Pescadillo and its yeast homolog (Yph1p or Nop7p) are localized to the nucleolus. Depletion of Nop7p leads to nuclear accumulation of pre-60S particles, indicating a defect in subunit export, and it interacts genetically with a tagged form of the ribosomal protein Rp125p, consistent with a role in subunit assembly. Two pre-rRNA processing pathways generate alternative forms of the 5.8S rRNA, designated 5-8S(L) and 5.8S(S). In cells depleted for Nop7p, the 27SA(3) pre-rRNA accumulated, whereas later processing intermediates and the mature 5.8S(S) rRNA were depleted. Less depletion was seen for the 5.8S(L) pathway. TAP-tagged Nop7p coprecipitated precursors to both 5.8S(L) and 5.8S(S) but not the mature rRNAs. We conclude that Nop7p is required for efficient exonucleolytic processing of the 27SA(3) pre-rRNA and has additional functions in 60S subunit assembly and transport. Nop7p is a component of at least three different pre-60S particles, and we propose that it carries out distinct functions in each of these complexes.

Ssf1p and Ssf2p are two nearly identical and functionally redundant nucleolar proteins. In the absence of Ssf1p and Ssf2p, the 27SA 2 pre-rRNA was prematurely cleaved, inhibiting synthesis of the 27SB and 7S pre-rRNAs and the 5.8S and 25S rRNA components of the large ribosomal subunit. On sucrose gradients, Ssf1p sedimented with pre-60S ribosomal particles. The 27SA 2 , 27SA 3 , and 27SB pre-rRNAs were copurified with tagged Ssf1p, as were 23 large subunit ribosomal proteins and 21 other proteins implicated in ribosome biogenesis. These included four Brix family proteins, Ssf1p, Rpf1p, Rpf2p, and Brx1p, indicating that the entire family functions in ribosome synthesis. This complex is distinct from recently reported pre-60S complexes in RNA and protein composition. We describe a multistep pathway of 60S preribosome maturation.

During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5′-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes., Author Summary R-loops (RNA-DNA hybrids) are potentially deleterious for gene expression and genome stability, but can be beneficial, for example, during immunoglobulin gene class-switch recombination. Here we made use of antibody S9.6, with specificity for RNA-DNA duplexes independently of their sequence. The genome-wide distribution of R-loops in wild-type yeast showed association with the highly transcribed ribosomal DNA, and protein-coding genes, particularly the second exon of spliced genes. On RNA polymerase III loci such as the highly transcribed transfer RNA genes (tRNAs), R-loop accumulation was strongly detected in the absence of both ribonucleases H1 and H2 (RNase H1 and H2), indicating that R-loops are inherently formed but rapidly cleared by RNase H. Importantly, stable R-loops lead to reduced synthesis of tRNA precursors in mutants lacking RNase H and DNA topoisomerase activities. RNA-DNA hybrids associated with TY1 cDNA retrotransposition intermediates were elevated in the absence of RNase H, and this was accompanied by increased retrotransposition, in particular to 5′-flanking regions of tRNAs. Our findings show that RNase H participates in silencing of TY1 life cycle. Surprisingly, R-loops associated with mitochondrial transcription units were suppressed specifically by RNase H1. These findings have potentially important implications for understanding human diseases caused by mutations in RNase H.

Summary Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2−/−Fanca−/− embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2., Graphical Abstract, Highlights • Maternal aldehydes must be catabolized to prevent fetal DNA damage • Fetal DNA repair counteracts DNA damage caused by maternal aldehydes • Aldh2−/−Rev1−/− mice do not phenocopy Aldh2−/−Fanca−/− mice • Maternal and fetal endogenous aldehydes corrupt development and hematopoiesis, During pregnancy, both mother and embryo produce reactive metabolites that can damage DNA. Oberbeck et al. find that there are distinct spatial and temporal roles for maternal and fetal aldehyde detoxification in protecting the developing embryo from DNA damage, and thus from accumulating developmental defects.

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles., Author Summary Recent progress has provided us with detailed knowledge of the structure and function of eukaryotic ribosomes. However, our understanding of the intricate processes of pre-ribosome assembly and the transition to translation-competent ribosomal subunits remains incomplete. The early and intermediate steps of ribosome assembly occur successively in the nucleolus and nucleoplasm. The pre-ribosomal subunits are then exported to the cytoplasm where final maturation steps, notably including D site cleavage of the 20S pre-rRNA to mature 18S rRNA, confer subunit joining and translation competence. Recent evidence indicates that pre-40S subunits are subject to a quality control step involving the GTP-dependent translation initiation factor eIF5B/Fun12, in the context of 80S-like ribosomes. Here, we demonstrate the involvement of 60S subunits in promoting 20S pre-rRNA cleavage. In particular, we show that a specific point mutation in the 60S subunit ribosomal protein L3 (rpl3[W255C]) leads to the accumulation of pre-40S particles that contain the 20S pre-rRNA but are translation-competent. Notably, this mutation prevents the stimulation of the GTPase activity of eIF5B/Fun12, which is also required for site D cleavage. We conclude that L3 plays an important role in regulating the function of eIF5B/Fun12 during 3′ end processing of 18S rRNA at site D, in the context of 80S ribosomes that have not yet engaged in translation.

When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells. DOI: http://dx.doi.org/10.7554/eLife.01641.001, eLife digest The genetic information of an organism is found in the nucleus of each cell in the form of DNA organised into chromosomes. The exact structure of those chromosomes changes as the cell moves through the different stages of the cell division cycle. During the stage called mitosis, where the DNA of a cell (which has previously been duplicated) is shared into two daughter cells, the chromosomes become tightly packed structures that can be readily moved through the cytoplasm. Since the late nineteenth century, it has been known that a layer of proteins, called the perichromosomal layer, coats the condensed chromosomes. However, virtually nothing was known about the role this layer performs. One of the first proteins to join the perichromosomal layer after mitosis begins is called Ki-67. This is only found in the cell nucleus when a cell is actively growing and dividing, and so is widely used as a marker in experiments investigating these processes: for example, Ki-67 is used to detect growing tumour cells amongst the normal cells in tissues of the body, and to measure the effectiveness of drugs designed to stop the growth of tumours. Again, however, little is known about what Ki-67 actually does. Booth et al. now reveal that when Ki-67 is not present in a cell, chromosomes do not have a perichromosomal layer—or at best, have a small remnant of one. This allowed Booth et al. to investigate the role of the perichromosomal layer as well. When the chromosomes first go through mitosis without a perichromosomal layer, no changes to the shape or the behaviour of the chromosomes are seen. However, the new nuclei are smaller than normal and their contents are arranged differently. This causes problems with the ability of daughter cells to synthesise protein building blocks and leads to an increased rate of spontaneous cell death when daughter cells try to undergo the next mitosis. Further research is needed to understand why this happens. DOI: http://dx.doi.org/10.7554/eLife.01641.002