Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres (CENs) via histone H3 variant CENP-A and low levels of CEN transcripts. RNA polymerase II (RNAPII) activity is restrained by the CEN histone code, while CEN RNA concentrations are reduced by the nuclear exosome. Using S. cerevisiae, we add kinase Rio1 to this scheme as it downregulates RNAPII, and promotes CEN RNA turnover via exoribonuclease Rat1. Transcription factor Cbf1 and the assembled kinetochore further restrain CEN transcription. CEN transcripts exist as long (up to 11,000nt) and short RNAs (119±40nt), which may underlie CEN identity and kinetochore recruitment. While also curtailed by Rio1, Rat1, and the exosome, periCEN RNAs (<200nt) accumulate at levels that are one order of magnitude higher than the CEN transcripts. Depleting Rio1 causes CEN and periCEN RNA buildup, kinetochore malformation, and chromosome loss. Depleting human orthologue RioK1 leads to CEN RNA accumulation and micronuclei formation, suggesting that Rio1/RioK1 activity at centromeres is conserved.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0–100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3’ and the 5’ splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

A shape-shifting nuclease unravels structured RNA

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3′-5′ exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3′-uridylated RNAs1-5. While the basis for Dis3L2 substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 Å. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.

NUDT2 initiates viral RNA degradation by removal of 5′-phosphates

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5′-triphosphate (PPP-) group that impairs degradation by the canonical 5′-3′ degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5′-3′ exonuclease XRN1. NUDT2 removes 5′-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5′-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.

Human 5p-tailed Mirtrons are Processed by RNaseP

Approximately a thousand microRNAs (miRNAs) are documented from human cells. A third appear to transit non-canonical pathways that typically bypass processing by Drosha, the dedicated nuclear miRNA producing enzyme. The largest class of non-canonical miRNAs are mirtrons which eschew Drosha to mature through spliceosome activity. While mirtrons are found in several configurations, the vast majority of human mirtron species are 5p-tailed. For these mirtrons, a 3p splice site defines the 3p end of their hairpin precursor while a tail of variable length separates the 5p base of the hairpin from the nearest splice site. How this tail is removed is not understood. Here we examine sequence motifs in 5p-tailed mirtrons and interactions with RNA turnover processes to characterize biogenesis processes. Through studying the high confidence 5p-tailed mirtron, hsa-miR-5010, we identify RNaseP as necessary and sufficient for severing the 5p tail of this mirtron. Further, depletion of RNaseP activity globally decreased 5p-tailed mirtron expression implicating this endoribonuclease in biogenesis of the entire class. Moreover, as 5p-tailed mirtron biogenesis appears to be connected to tRNA processing we found a strong correlation between accumulation of tRNA fragments (tRFs) and 5p-tailed mirtron abundance. This suggests that dysregulation of tRNA processing seen in cancers may also impact expression of the ~400 5p-tailed mirtrons encoded in the human genome.

Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner

SUMMARYLinker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we have identified a new function of histone H1 in chromatin structure and transcription using a range of genomic approaches. We show that histone H1-depleted cells accumulate nascent non-coding RNAs on chromatin, suggesting that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accumulated non-coding transcripts have reduced levels of m6A modification and cause replication-transcription conflicts. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism and DNA replication to maintain genome performance.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50µM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3’ and the 5’ splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 µM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

Quantitative Control for Stoichiometric Protein Synthesis

Bacterial protein synthesis rates have evolved to maintain preferred stoichiometries at striking precision, from the components of protein complexes to constituents of entire pathways. Setting relative protein production rates to be well within a factor of two requires concerted tuning of transcription, RNA turnover, and translation, allowing many potential regulatory strategies to achieve the preferred output. The last decade has seen a greatly expanded capacity for precise interrogation of each step of the central dogma genome-wide. Here, we summarize how these technologies have shaped the current understanding of diverse bacterial regulatory architectures underpinning stoichiometric protein synthesis. We focus on the emerging expanded view of bacterial operons, which encode diverse primary and secondary mRNA structures for tuning protein stoichiometry. Emphasis is placed on how quantitative tuning is achieved. We discuss the challenges and open questions in the application of quantitative, genome-wide methodologies to the problem of precise protein production. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover

Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number &gt;17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or species used in industrial applications. Bioinformatic analyses revealed similarity of the DUF1127 domain of bacterial proteins to the RNA binding domain of eukaryotic Smaug proteins that are involved in RNA turnover and have a role in development from Drosophila to mammals. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in maturation of the CcsR sRNAs that are processed from the 3′ UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 binds to many cellular RNAs of different type, several mRNAs with a function in cysteine / methionine / sulfur metabolism. It affects the stability of the CcsR RNAs and other non-coding RNAs and mRNAs. Thus, the widely distributed DUF1127 domain can mediate RNA-binding, affect stability of its binding partners and consequently modulate the bacterial transcriptome, thereby influencing different physiological processes.

S-Like Ribonuclease T2 Genes Are Induced during Mobilisation of Nutrients in Cotyledons from Common Bean

Germination and seedling development are crucial phases in a plant’s life cycle with economical and agronomical implications. The RNA quality in seeds is linked to seed viability, being an important agronomic trait since this leads to a loss in germination efficiency. In addition, RNA can be an important phosphorous reservoir in seeds, affecting the efficiency of the mobilisation of nutrients towards the seedlings. However, knowledge about the physiological function of ribonucleases during germination and seedling development is scarce. We analysed the ribonuclease activities of cotyledons during these processes and the expression of S-like ribonucleases T2. Ribonuclease activity was detected in cotyledons at 1 day after imbibition and the specific activity increased during germination and seedling development, reaching a maximal value at 10 days after imbibition. At this stage, the levels of proteins and RNA in cotyledons were very low. Using in-gel assays, three ribonucleases were detected with apparent molecular masses of 16, 17 and 19 kDa along cotyledon ontogeny. The S-like ribonucleases T2 family consists of four genes in common bean (PvRNS1 to PvRNS4). The expression of PvRNS1, PvRNS2 and PvRNS4 increased in the phase of nutrient mobilisation in cotyledons. The expression of PvRNS1 increased 1000 fold in cotyledons, from 1 to 6 days after imbibition. The suppression of the induction of ribonuclease activity and gene expression in decapitated seedlings suggests that the regulatory signal comes from the developing axes. These results clearly state that S-like ribonucleases T2 are involved in RNA turnover in cotyledons during seedling development.