Ribosomal Precursor RNA Research Articles

Atypical U3 snoRNA Suppresses the Process of Pterygium Through Modulating 18S Ribosomal RNA Synthesis.

BackgroundThe progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development.MethodsPrimary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis.ResultsReduced U3 snoRNA in pterygium promotes HCF or HPF cells’ proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5′ external transcribed spacer (5′ ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells.ConclusionsThe atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.

Discovery and Evaluation of Novel Angular Fused Pyridoquinazolinonecarboxamides as RNA Polymerase I Inhibitors.

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) into the 47S ribosomal RNA (rRNA) precursor. Further processing produces the 28S, 5.8S, and 18S rRNAs that are assembled into mature ribosomes. Many cancers exhibit higher Pol I transcriptional activity, reflecting a need for increased ribosome biogenesis and protein synthesis and making the inhibition of this process an attractive therapeutic strategy. Lead molecule BMH-21 (1) has been established as a Pol I inhibitor by affecting the destruction of RPA194, the Pol I large catalytic subunit. A previous structure-activity relationship (SAR) study uncovered key pharmacophores, but activity was constrained within a tight chemical space. This work details further SAR efforts that have yielded new scaffolds and improved off-target activity while retaining the desired RPA194 degradation potency. Pharmacokinetic profiling was obtained and provides a starting point for further optimization. New compounds present additional opportunities for the development of Pol I inhibitory cancer therapies.

9-Aminoacridine Inhibits Ribosome Biogenesis by Targeting Both Transcription and Processing of Ribosomal RNA.

Aminoacridines, used for decades as antiseptic and antiparasitic agents, are prospective candidates for therapeutic repurposing and new drug development. Although the mechanisms behind their biological effects are not fully elucidated, they are most often attributed to the acridines’ ability to intercalate into DNA. Here, we characterized the effects of 9-aminoacridine (9AA) on pre-rRNA metabolism in cultured mammalian cells. Our results demonstrate that 9AA inhibits both transcription of the ribosomal RNA precursors (pre-rRNA) and processing of the already synthesized pre-rRNAs, thereby rapidly abolishing ribosome biogenesis. Using a fluorescent intercalator displacement assay, we further show that 9AA can bind to RNA in vitro, which likely contributes to its ability to inhibit post-transcriptional steps in pre-rRNA maturation. These findings extend the arsenal of small-molecule compounds that can be used to block ribosome biogenesis in mammalian cells and have implications for the pharmacological development of new ribosome biogenesis inhibitors.

Identification of factors involved in ribosome assembly in the protozoan parasite Leishmania major

Formation of the ribosome subunits is a complex and progressive cellular process that requires a plethora of non-ribosomal transient proteins and diverse small nucleolar RNAs, which are involved from the synthesis of the precursor ribosomal RNA in the nucleolus to the final ribosome processing steps in the cytoplasm. Employing PTP-tagged Nop56 as a fishing bait to capture pre-ribosomal particles by tandem affinity purifications, mass spectrometry assays and a robust in silico analysis, here we describe tens of ribosome assembly factors involved in the synthesis of both ribosomal subunits in the human pathogen Leishmania major, where the knowledge about ribosomal biogenesis is scarce. We identified a large number of proteins that participate in most stages of ribosome biogenesis in yeast and mammals. Among them, we found several putative orthologs of factors not previously identified in L. major, such as t-Utp4, t-Utp5, Rrp7, Nop9 and Nop15. Even more interesting is the fact that we identified several novel candidates that could participate in the assembly of the atypical 60S subunit in L. major, which contains eight different rRNA species. As these proteins do not seem to have a human counterpart, they have potential as targets for novel anti-leishmanial drugs. Also, numerous proteins whose function is not apparently linked to ribosome assembly were copurified, suggesting that the L. major nucleolus is a multifunctional nuclear body.

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

Small nucleolar RNA is potential as a novel player in leukemogenesis and clinical application.

Lack of clarity of the mechanisms that underlie leukemogenesis obstructs the diagnosis, prognosis, and treatment of leukemia. Research has found that small nuclear RNA (snoRNA) plays an essential role in leukemia. These small non-coding RNAs are involved in ribosome biogenesis, including the 2′-O-methylation and pseudouridylation of precursor ribosomal RNA (pre-rRNA), and pre-rRNA splicing. Recently, many snoRNAs were found to be orphans that have no predictable RNA modification targets, but these RNAs have always been found to be located in different subcellular organelles, and they play diverse roles. Using high-throughput technology, snoRNA expression profiles have been revealed in leukemia, and some of the deregulated snoRNAs may regulate the cell cycle, differentiation, proliferation, and apoptosis in leukemic cells and confer drug resistance during leukemia treatment. In this review, we discuss the expression profiles and functions of snoRNAs, particularly orphan snoRNAs, in leukemia. It is possible that the dysregulated snoRNAs are promising diagnosis and prognosis markers for leukemia, which may serve as potential therapeutic targets in leukemia treatment.

Uncovering a delicate balance between endonuclease RNase III and ribosomal protein S15 in E. coli ribosome assembly

The bacterial ribosomal protein S15 is located in the platform, a functional region of the 30S ribosomal subunit. While S15 is critical for in vitro formation of E. coli small subunits (SSUs), it is dispensable for in vivo biogenesis and growth. In this work, a novel synergistic interaction between rpsO, the gene that encodes S15, and rnc (the gene that encodes RNase III), was uncovered in E. coli. RNase III catalyzes processing of precursor ribosomal RNA (rRNA) transcripts and thus is involved in functional ribosome subunit maturation. Strains lacking S15 (ΔrpsO), RNase III (Δrnc) or both genes were examined to understand the relationship between these two factors and the impact of this double deletion on rRNA processing and SSU maturation. The double deletion of rpsO and rnc partially alleviates the observed cold sensitivity of ΔrpsO alone. A novel 16S rRNA precursor (17S∗ rRNA) that is detected in free 30S subunits of Δrnc is incorporated in 70S-like ribosomes in the double deletion. The stable accumulation of 17S∗ rRNA suggests that timing of processing events is closely coupled with SSU formation events in vivo. The double deletion has a suppressive effect on the cell elongation phenotype of ΔrpsO. The alteration of the phenotypes associated with S15 loss, due to the absence of RNase III, indicates that pre-rRNA processing and improvement of growth, relative to that observed for ΔrpsO, are connected. The characterization of the functional link between the two factors illustrates that there are redundancies and compensatory pathways for SSU maturation.

The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-Dependent RNA Helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

The enigmatic RNase MRP of kinetoplastids

ABSTRACT The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

Steady-State Pre-rRNA Analysis to Investigate the Functional Microbiome.

The gut microbiome is recognized as a critical regulator of human diseases. Constituents of the microbiota and their individual activities can affect a broad range of disease states related to autoimmunity, cancer, infection, metabolism, mental health, and toxicant exposure. A substantial number of microbiome species are not culturable, limiting their study in vitro. Sequencing methods have allowed quantification of the composition of the microbiome, but methods to characterize the physiological status of bacterial species remain limited. Ribosomal RNA precursors (pre-rRNAs) are species-specific intermediates in bacterial ribosomal synthesis, and their levels are highly responsive to environmental changes. Immediately before and during active growth, pre-rRNA levels are high, whereas in non-dividing cells, copy numbers are orders of magnitude lower. These dynamics are conserved in all bacterial species and occur exclusively in viable cells, allowing the specific characterization of living and functional bacteria in their native states. Pre-rRNA analysis has been shown to yield valuable real-time information on the physiology of individual bacterial species within complex samples, beyond what traditional qPCR and sequencing methods can offer. Herein, we describe a PCR-based protocol to interrogate and quantify the in situ growth status of bacterial species of interest within a complex microbiome. We also describe an in vitro protocol to characterize the pre-rRNA/growth relationship for a given bacterial species to provide greater context for values obtained from natural samples. Improved understanding of microbial physiological responses to exposures could reveal novel toxicological mechanisms, biomarkers, and potential treatments. © 2021 Wiley Periodicals LLC. Basic Protocol: Targeted steady-state pre-rRNA analysis Support Protocol: Characterization of pre-rRNA/growth relationship © 2021 by John Wiley & Sons, Inc.

Spheres of Influence: Host Tree Proximity and Soil Chemistry Shape rRNA, but Not DNA, Communities of Symbiotic and Free-Living Soil Fungi in a Mixed Hardwood-Conifer Forest

Host and symbiont diversity are inextricably linked across partnerships and ecosystems, with degree of partner reliance governing the strength of this correlation. In many forest soils, symbiotic ectomycorrhizal fungi coexist and compete with free-living saprotrophic fungi, with the outcomes of these interactions shaping resource availability and competitive outcomes for the trees aboveground. Traditional approaches to characterizing these communities rely on DNA sequencing of a ribosomal precursor RNA gene (the internal transcribed spacer region), but directly sequencing the precursor rRNA may provide a more functionally relevant perspective on the potentially active fungal communities. Here, we map ectomycorrhizal and saprotrophic soil fungal communities through a mixed hardwood-conifer forest to assess how above- and belowground diversity linkages compare across these differently adapted guilds. Using highly spatially resolved transects (sampled every 2 m) and well-mapped stands of varying host tree diversity, we sought to understand the relative influence of symbiosis versus environment in predicting fungal diversity measures. Canopy species in this forest included two oaks (Quercus agrifoliaandQuercus douglasii) and one pine (Pinus sabiniana). At the scale of our study, spatial turnover in rRNA-based communities was much more predictable from measurable environmental attributes than DNA-based communities. And while turnover of ectomycorrhizal fungi and saprotrophs were predictable by the presence and abundance of different canopy species, they both responded strongly to soil nutrient characteristics, namely pH and nitrogen availability, highlighting the niche overlap of these coexisting guilds and the strong influence of aboveground plants on belowground fungal communities.

Conserved strategies of RNA polymerase I hibernation and activation

RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms ‘activation by cleft contraction’ and ‘hibernation by dimerization’ are unique to the regulation of this enzyme, but structure-function analysis is limited to baker’s yeast. To understand whether regulation by such strategies is specific to this model organism or conserved among species, we solve three cryo-EM structures of Pol I from Schizosaccharomyces pombe in different functional states. Comparative analysis of structural models derived from high-resolution reconstructions shows that activation is accomplished by a conserved contraction of the active center cleft. In contrast to current beliefs, we find that dimerization of the S. pombe polymerase is also possible. This dimerization is achieved independent of the ‘connector’ domain but relies on two previously undescribed interfaces. Our analyses highlight the divergent nature of Pol I transcription systems from their counterparts and suggest conservation of regulatory mechanisms among organisms.

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2.

RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels in balance with PABPs and other RBPs with mutable substrate specificity across nucleoplasmic and nucleolar RNA processes.

Loss of POLR1D results in embryonic lethality prior to blastocyst formation in mice.

In eukaryotic cells, RNA polymerase (Pol) I and Pol III are dedicated to the synthesis of ribosomal RNA precursors and a variety of small RNAs, respectively. Although RNA Pol I and Pol III complexes are crucial for the regulation of cell growth and cell cycle in all cell types, many of the components of the Pol I and Pol III complexes have not been functionally characterized in mammals. Here, we provide the first in vivo functional characterization of POLR1D, a subunit shared by RNA Pol I and Pol III, during early mammalian embryo development. Our results show that Polr1d mutant embryos cannot be recovered at E7.5 early post-gastrulation stage, suggesting failed implantation. Although Polr1d mutants can be recovered at E3.5, they exhibit delayed/stalled development with morula morphology rather than differentiation into blastocysts. Even with extended time in culture, mutant embryos fail to form blastocysts and eventually die. Analysis of E3.0 embryos revealed severe DNA damage in Polr1d mutants. Additionally, lineage assessment reveals that trophectoderm specification is compromised in the absence of Polr1d. In summary, these findings demonstrate the essential role of POLR1D during early mammalian embryogenesis and highlight cell-lethal phenotype without Polr1d function.

Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.

Staufen1 localizes to the mitotic spindle and controls the localization of RNA populations to the spindle.

Staufen1 (STAU1) is an RNA-binding protein involved in the post-transcriptional regulation of mRNAs. We report that a large fraction of STAU1 localizes to the mitotic spindle in colorectal cancer HCT116 cells and in non-transformed hTERT-RPE1 cells. Spindle-associated STAU1 partly co-localizes with ribosomes and active sites of translation. We mapped the molecular determinant required for STAU1-spindle association within the first 88 N-terminal amino acids, a domain that is not required for RNA binding. Interestingly, transcriptomic analysis of purified mitotic spindles revealed that 1054 mRNAs and the precursor ribosomal RNA (pre-rRNA), as well as the long non-coding RNAs and small nucleolar RNAs involved in ribonucleoprotein assembly and processing, are enriched on spindles compared with cell extracts. STAU1 knockout causes displacement of the pre-rRNA and of 154 mRNAs coding for proteins involved in actin cytoskeleton organization and cell growth, highlighting a role for STAU1 in mRNA trafficking to spindle. These data demonstrate that STAU1 controls the localization of subpopulations of RNAs during mitosis and suggests a novel role of STAU1 in pre-rRNA maintenance during mitosis, ribogenesis and/or nucleoli reassembly.

Structural insight into precursor ribosomal RNA processing by ribonuclease MRP.

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the ‘core factor’ subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.

Full-length NF-κB repressing factor contains an XRN2 binding domain.

NF-κB repressing factor (NKRF) was recently identified as an RNA binding protein that together with its associated proteins, the 5′–3′ exonuclease XRN2 and the helicase DHX15, is required to process the precursor ribosomal RNA. XRN2 is a multi-functional ribonuclease that is also involved in processing mRNAs, tRNAs and lncRNAs. The activity and stability of XRN2 are controlled by its binding partners, PAXT-1, CDKN2AIP and CDKN2AIPNL. In each case, these proteins interact with XRN2 via an XRN2 binding domain (XTBD), the structure and mode of action of which is highly conserved. Rather surprisingly, although NKRF interacts directly with XRN2, it was not predicted to contain such a domain, and NKRF's interaction with XRN2 was therefore unexplained. We have identified an alternative upstream AUG start codon within the transcript that encodes NKRF and demonstrate that the full-length form of NKRF contains an XTBD that is conserved across species. Our data suggest that NKRF is tethered in the nucleolus by binding directly to rRNA and that the XTBD in the N-terminal extension of NKRF is essential for the retention of XRN2 in this sub-organelle. Thus, we propose NKRF regulates the early steps of pre-rRNA processing during ribosome biogenesis by controlling the spatial distribution of XRN2 and our data provide further support for the XTBD as an XRN2 interacting motif.

Expression and Clinical Significance of POLR1D in Colorectal Cancer

Purpose: RNA polymerase I subunit D (POLR1D) is involved in the synthesis of ribosomal RNA precursors and small RNAs, but its mechanism in the development and progression of colorectal cancer (CRC) remains ambiguous. Thus, this research aimed to investigate POLR1D’s expression and significance in human CRC patients and evaluate its association with clinicopathological characteristics. Methods: Matched fresh-frozen cancerous and non-cancerous tissues were collected from 100 patients diagnosed with CRC. Immunohistochemical, Western blot, and quantitative real-time polymerase chain reaction analyses were adopted to validate the correlation between POLR1D expression and clinicopathological parameters in CRC tissues and adjacent normal tissues (ANTs). Results: POLR1D expression in CRC tissues was significantly higher than in the ANTs. χ² test and Spearman’s correlative analysis showed that a high POLR1D expression is significantly associated with clinical stage, Dukes stage, tumor differentiation, depth of invasion, and metastasis (p < 0.05). It is not correlated with gender, age, and tumor location and size (p > 0.05). Kaplan-Meier survival curves show that the overall survival (OS) time for the low expression group is remarkably longer than for the high expression group (p < 0.0015). Univariate and multivariate analyses indicate that a high POLR1D expression is an independent prognostic factor for poor OS (p = 0.000). Conclusion: The findings of this study strongly indicate that POLR1D plays a pivotal role in the occurrence and progression of CRC. It might be an independent adverse prognostic factor for CRC patients and could serve as a potential therapeutic target for clinical diagnosis in CRC and anticancer drug development.