Ribosomal Precursor RNA Research Articles

Localization study of CHD7 protein expressed in HeLa and HEK293 cells

CHARGE syndrome is a rare genetic disease characterized by numerous congenital abnormalities caused by de novo alterations of the CHD7 gene. It encodes a chromo domain protein, involved in the ATP-dependent remodelling of chromatin. It has been described that CHD7 protein plays a role as a positive regulator of the nucleolar expression of the 45S ribosomal RNA precursor. The aim of this research was to study where the CHD7 protein is localized. Knowledge of the subcellular localization of the CHD7 protein is essential for understanding its physiological function. This study was conducted by transfection of two plasmids encoding CHD7-HA or FLAG-CHD7 into HeLa (human cervix carcinoma) and HEK293 (human embryonic kidney) cells. Protein expression and localization of the CHD7 protein was demonstrated by western blot and immunofluorescence. CHD7 protein was expressed in both cell models, and localization of the protein was found in the nucleoplasm, but not in the nucleolus.

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 poly-nucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA. 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-electron microscopy 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.

Nol9 Is a Spatial Regulator for the Human ITS2 Pre-rRNA Endonuclease–Kinase Complex

The ribosome plays a universal role in translating the cellular proteome. Defects in the ribosome assembly factor Las1L are associated with congenital lethal motor neuron disease and X-linked intellectual disability disorders, yet its role in processing precursor ribosomal RNA (pre-rRNA) is largely unclear. The Las1L endoribonuclease associates with the Nol9 polynucleotide kinase to form the internal transcribed spacer 2 (ITS2) pre-rRNA endonuclease–kinase machinery. Together, Las1L–Nol9 catalyzes RNA cleavage and phosphorylation to mark the ITS2 for degradation. While ITS2 processing is critical for the production of functional ribosomes, the regulation of mammalian Las1L–Nol9 remains obscure. Here we characterize the human Las1L–Nol9 complex and identify critical molecular features that regulate its assembly and spatial organization. We establish that Las1L and Nol9 form a higher-order complex and identify the regions responsible for orchestrating this intricate architecture. Structural analysis by high-resolution imaging defines the intricate spatial pattern of Las1L–Nol9 within the nucleolar sub-structure linked with late pre-rRNA processing events. Furthermore, we uncover a Nol9-encoded nucleolar localization sequence that is responsible for nucleolar transport of the assembled Las1L–Nol9 complex. Together, these data provide a mechanism for the assembly and nucleolar localization of the human ITS2 pre-rRNA endonuclease–kinase complex.

Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I.

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.

POLR1D promotes colorectal cancer progression and predicts poor prognosis of patients.

RNA polymerase I subunit D (POLR1D), which is involved in synthesis of ribosomal RNA precursors and small RNAs, has been shown to be overexpressed in several human cancer types. Nevertheless, the role of POLR1D in the progression of colorectal cancer (CRC) remains unknown. The following study aimed to investigate the role and underlying mechanism of POLR1D in CRC progression. In this report, we found that POLR1D was significantly up-regulated in CRC through data mining of oncomine database. Furthermore, the immunohistochemistry (IHC) staining of a tissue microarray (TMA) of 75 human CRC patients showed that the expression level of POLR1D was positively correlated to tumor size and poor survival of CRC patients. Aberrant expression of POLR1D significantly promoted cell proliferation and migration in vitro, as well as tumor growth in vivo. Conversely, POLR1D knockdown displayed the opposite effects. The flow Cytometry assays showed that POLR1D fostered cell cycle progression at G1-S transition and inhibited cell apoptosis. Finally, at the molecular level, we demonstrated that POLR1D-induced the promotion of G1-S cell cycle transition was mediated by activation of wnt-β-catenin signaling and inactivation of p53 signaling. Our results suggested that POLR1D may function as a risk factor for predicting the outcome of CRC patients, as well as a potential therapeutic target for CRC.

Linking RNA Processing and Function.

RNA processing is critical for eukaryotic mRNA maturation and function. It appears there is no exception for other types of RNAs. Long noncoding RNAs (lncRNAs) represent a subclass of noncoding RNAs, have sizes of >200 nucleotides (nt), and participate in various aspects of gene regulation. Although many lncRNAs are capped, polyadenylated, and spliced just like mRNAs, others are derived from primary transcripts of RNA polymerase II and stabilized by forming circular structures or by ending with small nucleolar RNA-protein complexes. Here we summarize the recent progress in linking the processing and function of these unconventionally processed lncRNAs; we also discuss how directional RNA movement is achieved using the radial flux movement of nascent precursor ribosomal RNA (pre-rRNA) in the human nucleolus as an example.

Identification of the RNA polymerase I-RNA interactome.

Ribosome biogenesis is a complex process orchestrated by a host of ribosome assembly factors. Although it is known that many of the proteins involved in this process have RNA binding activity, the full repertoire of proteins that interact with the precursor ribosomal RNA is currently unknown. To gain a greater understanding of the extent to which RNA-protein interactions have the potential to control ribosome biogenesis, we used RNA affinity isolation coupled with proteomics to measure the changes in RNA-protein interactions that occur when rRNA transcription is blocked. Our analysis identified 211 out of 457 nuclear RNA binding proteins with a >3-fold decrease in RNA-protein interaction after inhibition of RNA polymerase I (RNAPI). We have designated these 211 RNA binding proteins as the RNAPI RNA interactome. As expected, the RNAPI RNA interactome is highly enriched for nucleolar proteins and proteins associated with ribosome biogenesis. Selected proteins from the interactome were shown to be nucleolar in location and to have RNA binding activity that was dependent on RNAPI activity. Furthermore, our data show that two proteins, which are required for rRNA maturation, AATF and NGDN, and which form part of the RNA interactome, both lack canonical RNA binding domains and yet are novel pre-rRNA binding proteins.

Structural basis of RNA polymerase I stalling at UV light-induced DNA damage

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

The RNA exosome captured in action

Structural Biology The RNA exosome, a major RNA degradation machine, processes ribosomal RNA (rRNA) precursors and is directly coupled to the protein synthesis machine, the ribosome. Using cryo–electron microscopy, Schuller et al. investigated the structure of the precursor large ribosomal subunit from yeast with unprocessed rRNA in complex with the RNA exosome. The structure captures a snapshot of two molecular machines transiently interacting and explains how the RNA exosome acts on an authentic physiological substrate and remodels the large subunit during ribosome maturation. Science , this issue p. [219][1] [1]: /lookup/doi/10.1126/science.aar5428

Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA.

The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. cerevisiae, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.

Enhanced skeletal muscle ribosome biogenesis, yet attenuated mTORC1 and ribosome biogenesis-related signalling, following short-term concurrent versus single-mode resistance training

Combining endurance training with resistance training (RT) may attenuate skeletal muscle hypertrophic adaptation versus RT alone; however, the underlying mechanisms are unclear. We investigated changes in markers of ribosome biogenesis, a process linked with skeletal muscle hypertrophy, following concurrent training versus RT alone. Twenty-three males underwent eight weeks of RT, either performed alone (RT group, n = 8), or combined with either high-intensity interval training (HIT+RT group, n = 8), or moderate-intensity continuous training (MICT+RT group, n = 7). Muscle samples (vastus lateralis) were obtained before training, and immediately before, 1 h and 3 h after the final training session. Training-induced changes in basal expression of the 45S ribosomal RNA (rRNA) precursor (45S pre-rRNA), and 5.8S and 28S mature rRNAs, were greater with concurrent training versus RT. However, during the final training session, RT further increased both mTORC1 (p70S6K1 and rps6 phosphorylation) and 45S pre-rRNA transcription-related signalling (TIF-1A and UBF phosphorylation) versus concurrent training. These data suggest that when performed in a training-accustomed state, RT induces further increases mTORC1 and ribosome biogenesis-related signalling in human skeletal muscle versus concurrent training; however, changes in ribosome biogenesis markers were more favourable following a period of short-term concurrent training versus RT performed alone.

Molecular viability testing of viable but non-culturable bacteria induced by antibiotic exposure.

SummaryNucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNC P. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells.

Structural insights into transcription initiation by yeast RNA polymerase I

In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre‐rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi‐subunit pre‐initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo‐EM structure of the 18‐subunit yeast Pol I PIC bound to a transcription scaffold. The cryo‐EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB‐related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a unique example of a protein-guided programmable endoribonuclease.

Novel Assay to Detect RNA Polymerase I Activity In Vivo.

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577-84. ©2017 AACR.

Box C/D small nucleolar RNA genes and the Prader‐Willi syndrome: a complex interplay

The nucleolus of mammalian cells contains hundreds of box C/D small nucleolar RNAs (SNORDs). Through their ability to base pair with ribosomal RNA precursors, most play important roles in the synthesis and/or activity of ribosomes, either by guiding sequence-specific 2'-O-methylations or by facilitating RNA folding and cleavages. A growing number of SNORD genes with elusive functions have been discovered recently. Intriguingly, the vast majority of them are located in two large, imprinted gene clusters at human chromosome region 15q11q13 (the SNURF-SNRPN domain) and at 14q32 (the DLK1-DIO3 domain) where they are expressed, respectively, only from the paternally and maternally inherited alleles. These placental mammal-specific SNORD genes have many features of the canonical SNORDs that guide 2'-O-methylations, yet they lack obvious complementarity with ribosomal RNAs and, surprisingly, they are processed from large, tandemly repeated genes expressed preferentially in the brain. This review summarizes our understanding of the biology of these peculiar SNORD genes, focusing particularly on SNORD115 and SNORD116 in the SNURF-SNRPN domain. It examines the growing evidence that altered levels of these SNORDs and/or their host-gene transcripts may be a primary cause of Prader-Willi syndrome (PWS; a rare disorder characterized by overeating and obesity) as well as abnormalities in signaling through the 5-HT2C serotonin receptor. Finally, the hypothesis that PWS may be a ribosomopathy (ribosomal disease) is also discussed. WIREs RNA 2017, 8:e1417. doi: 10.1002/wrna.1417 For further resources related to this article, please visit the WIREs website.

C-Myc co-ordinates mRNA cap methylation and ribosomal RNA production.

The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)–RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT–RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT–RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT–RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT–RAM is suppressed, indicating that RNMT–RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT–RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.

Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis.

Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth.

Biochemical properties of Bacillus Calmette Guerin ribonuclease III.

Double-stranded RNA (dsRNA) is discovered to participate in the regulation of gene expression in both bacterial and eukaryotic cells. Members of ribonuclease III (RNase III) family recognize RNA motifs and cleave substrates at specific sites in a divalent-metal-ion-dependent manner. In this study, we find the RNase III from Bacillus Calmette Guerin (BCG-RNase III) cleaves small hairpin RNA based on the conserved stem structure associated with Mycobacterium 16S ribosomal RNA precursor at specific sites which are not determined. To evaluate the influence of remnant endogenous ribonucleases from expression host on RNA cleavage assays for RNase III, we use E44A and D48A mutant of the enzyme to perform RNA cleavage assays and find that remnant ribonucleases have no effect on cleavage assays. The RNA cleavage activity of the enzyme can be supported by Mg(2+), Mn(2+), and Co(2+) and enhanced with the increasing salt concentration. The catalytic activity of the enzyme is exhibited when the temperature of the reaction buffer ranges from 30 to 55 °C and the pH of the buffer from 7.0 to 10.0. Two major cleavage sites in RNA substrate are identified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE).

An alternative RNA polymerase I structure reveals a dimer hinge.

RNA polymerase I (Pol I) is the central, 14-subunit enzyme that synthesizes the ribosomal RNA (rRNA) precursor in eukaryotic cells. The recent crystal structure of Pol I at 2.8 Å resolution revealed two novel elements: the `expander' in the active-centre cleft and the `connector' that mediates Pol I dimerization [Engel et al. (2013), Nature (London), 502, 650-655]. Here, a Pol I structure in an alternative crystal form that was solved by molecular replacement using the original atomic Pol I structure is reported. The resulting alternative structure lacks the expander but still shows an expanded active-centre cleft. The neighbouring Pol I monomers form a homodimer with a relative orientation distinct from that observed previously, establishing the connector as a hinge between Pol I monomers.