Mature rRNA Research Articles

The evolution of the Puf superfamily of proteins across the tree of eukaryotes

BackgroundEukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity.ResultsHere, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization.ConclusionsOur data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

Gapmer Antisense Oligonucleotides Targeting 5S Ribosomal RNA Can Reduce Mature 5S Ribosomal RNA by Two Mechanisms.

In this study, we demonstrate that 5S ribosomal RNA (rRNA), a highly structured and protein-bound RNA, is quite difficult to reduce with antisense oligonucleotides (ASOs). However, we found a single accessible site that was targetable with a high-affinity complementary ASO. The ASO appeared to bind to the site, recruit RNaseH1, and cause degradation of the 5S RNA. Intriguingly, we also observed that the same ASO induced an accumulation of pre-5S RNA, which may contribute to reduced levels of mature 5S rRNA. As expected, ASO mediated reduction of 5S RNA, and modest inhibition of processing of pre-5S RNA resulted in nucleolar toxicity. However, the toxicity induced was minimal compared with actinomycin D, consistent with its modest effects on pre-5S rRNA. Mechanistically, we show that the accumulation of pre-5S rRNA required ASO hybridization to the cognate rRNA sequence but was independent of RNaseH1 activity. We found that Ro60 and La, proteins known to bind misprocessed RNAs, likely sequester the ASO-pre-5S rRNA species and block RNaseH1 activity, thus identifying another example of competitive mechanisms mediated by proteins that compete with RNaseH1 for binding to ASO-RNA heteroduplexes.

Comprehensive analysis of the pre-ribosomal RNA maturation pathway in a methanoarchaeon exposes the conserved circularization and linearization mode in archaea

ABSTRACT The ribosomal RNA (rRNA) genes are generally organized as an operon and cotranscribed into a polycistronic precursor; therefore, processing and maturation of pre-rRNAs are essential for ribosome biogenesis. However, rRNA maturation pathways of archaea, particularly of methanoarchaea, are scarcely known. Here, we thoroughly elucidated the maturation pathway of the rRNA operon (16S-tRNAAla-23S-tRNACys-5S) in Methanolobus psychrophilus, one representative of methanoarchaea. Enzymatic assay demonstrated that EndA, a tRNA splicing endoribonuclease, cleaved bulge-helix-bulge (BHB) motifs buried in the processing stems of pre-16S and pre-23S rRNAs. Northern blot and quantitative PCR detected splicing-coupled circularization of pre-16S and pre-23S rRNAs, which accounted for 2% and 12% of the corresponding rRNAs, respectively. Importantly, endoribonuclease Nob1 was determined to linearize circular pre-16S rRNA at the mature 3′ end so to expose the anti-Shine–Dalgarno sequence, while circular pre-23S rRNA was linearized at the mature 5′ end by an unknown endoribonuclease. The resultant 5′ and 3′ extension in linearized pre-16S and pre-23S rRNAs were finally matured through 5′-3′ and 3′-5′ exoribonucleolytic trimming, respectively. Additionally, a novel processing pathway of endoribonucleolysis coupled with exoribonucleolysis was identified for the pre-5S rRNA maturation in this methanogen, which could be also conserved in most methanogenic euryarchaea. Based on evaluating the phylogenetic conservation of the key elements that are involved in circularization and linearization of pre-rRNA maturation, we predict that the rRNA maturation mode revealed here could be prevalent among archaea.

A newly identified duplex RNA unwinding activity of archaeal RNase J depends on processive exoribonucleolysis coupled steric occlusion by its structural archaeal loops

ABSTRACT RNase J is a prokaryotic 5′-3′ exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5′ single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.

Research advance of ANRIL on atherosclerosis by regulating cell proliferation and apoptosis

Atherosclerosis is an important pathological basis for coronary artery disease. ANRIL is an antisense non-coding RNA located in Chr9p21 locus, which was identified as the most significant risk locus associated with atherosclerosis. ANRIL can produce multiple transcripts including linear and circular transcripts after various transcript splicing. It has been illustrated that ANRIL plays important roles in the pathology of atherosclerosis by regulating the proliferation and apoptosis of vascular cells. Linear ANRIL can regulate the proliferation of vascular smooth muscle cells (VSMCs) in plaques by chromatin modification, as well as influence the proliferation and the apoptosis of macrophages in post transcription; circular ANRIL can affect the proliferation and apoptosis of VSMCs by chromatin modification as well as interfering with rRNA maturation. In this review, we describe the ANRIL evolution, different transcripts characteristics, and their roles in the proliferation and apoptosis of vascular cells to participate in the process of atherosclerosis, for further understanding the pathogenesis of atherosclerosis and finding potential targets for diagnosis and treatment of atherosclerosis.

RPL32 Promotes Lung Cancer Progression by Facilitating p53 Degradation

Lung cancer is the leading cause of cancer death worldwide, and the overall survival rate of advanced lung cancer patients is unsatisfactory. Ribosomal proteins (RPs) play important roles in carcinogenesis. However, the role of RPL32 in lung cancer has not been demonstrated. Here, we report that RPL32 is aberrantly, highly expressed in lung cancer tissues and that the overexpression of RPL32 is correlated with the poor prognosis of these patients. RPL32 silencing significantly inhibited the proliferation of lung cancer cells, with an observed p53 accumulation and cell-cycle arrest. Mechanistically, knockdown of RPL32 resulted in ribosomal stress and affected rRNA maturation. RPL5 and RPL11 sensed stress and translocated from the nucleus to the nucleoplasm, where they bound to murine double minute 2 (MDM2), an important p53 E3 ubiquitin ligase, which resulted in p53 accumulation and inhibition of cancer cell proliferation. As lung cancer cells usually express high levels of Toll-like receptor 9 (TLR9), we conjugated RPL32 small interfering RNA (siRNA) to the TLR9 ligand CpG to generate CpG-RPL32 siRNA, which could stabilize and guide RPL32 siRNA to lung cancer cells. Excitingly, CpG-RPL32 siRNA displayed strong anticancer abilities in lung cancer xenografts. Therefore, RPL32 is expected to be a potential target for lung cancer treatment.

The ribotoxin α-sarcin can cleave the sarcin/ricin loop on late 60S pre-ribosomes.

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.

The 18S ribosomal RNA m6 A methyltransferase Mettl5 is required for normal walking behavior in Drosophila.

RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6 A methyltransferases were identified, uncovering the specificity of m6 A deposition by structurally distinct enzymes. In order to discover additional m6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.

RNase AM, a 5' to 3' exonuclease, matures the 5' end of all three ribosomal RNAs in E. coli.

Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5′-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5′ to 3′ exonuclease, performs the last step of 5S rRNA 5′-end maturation. RNase AM was also found to generate the mature 5′ end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5′ end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5′ to 3′ RNA exonuclease identified in E. coli.

Modification of Adenosine196 by Mettl3 Methyltransferase in the 5'-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation.

Ribosome biogenesis is among the founding processes in the cell. During the first stages of ribosome biogenesis, polycistronic precursor of ribosomal RNA passes complex multistage maturation after transcription. Quality control of preribosomal RNA (pre-rRNA) processing is precisely regulated by non-ribosomal proteins and structural features of pre-rRNA molecules, including modified nucleotides. However, many participants of rRNA maturation are still unknown or poorly characterized. We report that RNA m6A methyltransferase Mettl3 interacts with the 5′ external transcribed spacer (5′ETS) of the 47S rRNA precursor and modifies adenosine 196. We demonstrated that Mettl3 knockdown results in the increase of pre-rRNA processing rates, while intracellular amounts of rRNA processing machinery components (U3, U8, U13, U14, and U17 small nucleolar RNA (snoRNA)and fibrillarin, nucleolin, Xrn2, and rrp9 proteins), rRNA degradation rates, and total amount of mature rRNA in the cell stay unchanged. Increased efficacy of pre-rRNA cleavage at A’ and A0 positions led to the decrease of 47S and 45S pre-rRNAs in the cell and increase of mature rRNA amount in the cytoplasm. The newly identified conserved motif DRACH sequence modified by Mettl3 in the 5′-ETS region is found and conserved only in primates, which may suggest participation of m6A196 in quality control of pre-rRNA processing at initial stages demanded by increased complexity of ribosome biogenesis.

Genetic Variation and Potential for Resistance Development to the tTA Overexpression Lethal System in Insects.

Release of insect pests carrying the dominant lethal tetracycline transactivator (tTA) overexpression system has been proposed as a means for population suppression. High levels of the tTA transcription factor are thought to be toxic due to either transcriptional squelching or interference with protein ubiquitination. Here we utilized the Drosophila melanogaster Genetic Reference Panel (DGRP) to examine the influence of genetic variation on the efficacy of a female-specific tTA overexpression system. The level of female lethality between DGRP lines varied from 11 to 97% with a broad sense heritability of 0.89. A genome-wide association analysis identified 192 allelic variants associated with high or low lethality (P < 10−5), although none were significant when corrected for multiple testing. 151 of the variants fell within 108 genes that were associated with several biological processes including transcription and protein ubiquitination. In four lines with high female lethality, tTA RNA levels were similar or higher than in the parental tTA overexpression strain. In two lines with low lethality, tTA levels were about two fold lower than in the parental strain. However, in two other lines with low lethality, tTA levels were similar or approximately 30% lower. RNAseq analysis identified genes that were up or downregulated in the four low female lethal lines compared to the four high lethal lines. For example, genes associated with RNA processing and rRNA maturation were significantly upregulated in low lethal lines. Our data suggest that standing genetic variation in an insect population could provide multiple mechanisms for resistance to the tTA overexpression system.

Impaired chondrcoyte U3 SNORNA expression in osteoarthritis and its impact on the chondrocyte's protein translation apparatus

Purpose: Osteoarthritis (OA) is the most prevalent degenerative joint disease with no disease-modifying treatment available. During OA progression, articular chondrocyte homeostasis is disturbed and presents with a catabolic signature. Pathways controlling ribosome activity have previously been described in the regulation of chondrocyte homeostasis. However, ribosome biogenesis has not been implicated in OA. We hypothesized that U3 small nucleolar RNA (snoRNA), a key factor in the endoribonucleolytic ribosomal RNA (rRNA) maturation, is critical for chondrocyte homeostasis via regulating the availability of mature rRNAs.

The 47S Pre-rRNA processing cascade of articular chondrocytes is impaired in an in vitro model of osteoarthritis

Purpose: Osteoarthritis (OA) is the most common degenerative joint disorder, characterized by progressive loss and destruction of articular cartilage. Chondrocytes are highly specialized cells with primary function of producing and maintaining the extracellular matrix (ECM). This requires continuous translation of mRNAs by ribosomes. Mammalian ribosomes are large protein-RNA complexes containing 79 protein subunits and 4 ribosomal RNAs (18S, 5.8S, 28S, and 5 rRNAs). In the human system, three ribosomal RNAs, 18S, 5.8S, and 28S, arise from a single 47S pre-rRNA transcript. Maturation of these rRNAs takes place by a cascade of exo- and endo-ribonucleolytic cleavages of the primary 47S transcript. A limited number of small nucleolar RNAs (snoRNAs), such as RMRP are known to guide endoribonucleolytic cleavages of the 47S pre-rRNA transcript. Here, we hypothesized that 47S pre-rRNA processing is impaired in osteoarthritic articular chondrocytes, and contributes to OA progression. Methods: Primary human knee OA articular chondrocytes (pool of four donors, p2, seeding density 30,000 cells/cm2) were cultured in DMEM/F12, 10% FCS, 1% P/S, 1% NEAA and exposed to knee OA synovial fluid (OA-SF, 20% (v/v), pool of five donors) for 1-14 days. Expression levels of chondrocytes phenotypic marker genes (COL2A1, ACAN, COL10A1, COL1A1), selected 47S processing intermediates and RMRP snoRNA were measured by qPCR. Statistical significance was assessed by two-way ANOVA, Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 Results: To test our hypothesis, we set-up an OA-mimicking in vitro model using human primary articular chondrocytes exposed to osteoarthritic SF (OA-SF) for 1 to 14 days. The expression of chondrogenic marker genes COL2A1 (Figure 1A) and ACAN, as well as hypertrophic marker gene COL10A1, were significantly decreased immediately after the first day of treatment with OA-SF. The expression of COL1A1 (Figure. 1B), a marker of chondrocyte dedifferentiation, increased significantly upon OA-SF treatment in a time-dependent manner. To evaluate the 47S pre-rRNA processing cascade, primers covering selected cleavage sites (01, 2 and 4a) in the 47S transcript were designed (Figure 1C). Upon OA-SF treatment, we observed accelerated cleavage at site 01 within the 5’ ETS region, and site 2 within the ITS1. Cleavage at site 2 separates pre-18S and pre-5.8S-28S intermediates and it is guided by snoRNA RMRP, expression of which was increased upon OA-SF treatment. On the contrary, cleavage at site 4a within the ITS2 region, generating separated pre-5.8S and pre-28S precursors, was hindered in our model (Figure 1D). These results demonstrate that the dynamics of the 47S primary transcript processing is altered in our OA-mimicking in vitro model, showing accelerated processing towards mature 18S rRNA, and hindered processing towards mature 5.8S and 28S rRNAs. Conclusions: During OA, the chondrocyte’s environment changes dramatically, fueling a gradual change of the chondrocyte’s phenotype. In response to OA onset and progression, chondrocytes can adopt a variety of different OA-associated phenotypes ranging from proliferative, pre-hypertrophic, hypertrophic to a fibroblast-like. Upon OA-SF treatment we observed a progressive decrease in expression of COL2A1, ACAN, as well as COL10A1 genes, while expression of the dedifferentiation marker gene COL1A1 was significantly increased. Such an expression pattern is typical for dedifferentiating chondrocytes, and it indicates that chondrocytes treated with OA-SF are predominantly acquiring a fibroblast-like phenotype. Our results demonstrate alterations in the 47S pre-rRNA processing cascade, suggesting that 47S processing is vulnerable to changes in the chondrocyte’s extracellular environment. That can potentially lead to OA pathobiologically important alterations in ribosome biogenesis with consequences for the chondrocyte’s proteome. Future experiments are focussing on the validation of these findings by northern blotting and detailed unbiased mapping of the 47S processing cascade using RNA sequencing technology.

Functional regions in the 5' external transcribed spacer of yeast pre-rRNA.

Ribosomal subunits are assembled on a precursor rRNA that includes four spacers in addition to mature rRNA sequences. The 5′ external transcribed spacer (5′ ETS) is the most prominent one that recruits U3 snoRNA and a plethora of proteins during the early assembly of 90S small subunit preribosomes. Here, we have conducted a comprehensive mutational analysis of 5′ ETS by monitoring the processing and assembly of a plasmid-expressed pre-18S RNA. Remarkably, nearly half of the 5′ ETS sequences, when depleted individually, are dispensable for 18S rRNA processing. The dispensable elements largely bind at the surface of the 90S structure. Defective assembly of 5′ ETS completely blocks the last stage of 90S formation yet has little effect on the early assembly of 5′ and central domains of 18S rRNA. Our study reveals the functional regions of 5′ ETS and provides new insight into the assembly hierarchy of 90S preribosomes.

Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitoribosomal RNA of the minor subunit, 15S rRNA, is transcribed as a bicistronic transcript along with tRNAW. 5' and 3' sequences flanking the mature transcript must be removed by cleavage at the respective junctions before incorporating it into the mitoribosome. An in vivo dose-response triphasic system was created to elucidate the role of Ccm1p in the processing of 15S rRNA: Ccm1p supply ("On"), deprivation ("Off"), and resupply ("Back on"). After 72h under "Off" status, the cells started to exhibit a complete mutant phenotype as assessed by their lack of growth in glycerol medium, while keeping their mitochondrial DNA integrity (ρ+). Full functionality of mitochondria was reacquired upon "Back on." 15S rRNA levels and phenotype followed the Ccm1p intramitochondrial concentrations throughout the "On-Off-Back on" course. Under "Off" status, cells gradually accumulated unprocessed 5' and 3' junctions, which reached significant levels at 72-96h, probably due to a saturation of the mitochondrial degradosome (mtEXO). The Ccm1p/mtEXO mutant (Δccm1/Δdss1) showed a copious accumulation of 15S rRNA primary transcript forms, which were cleaved upon Ccm1p resupply. The gene that codes for the RNA component of RNase P was conserved in wild-type and mutant strains. Our results indicate that Ccm1p is crucial in processing the 15S rRNA primary transcript and does not stabilize the already mature 15S rRNA. Consequently, failure of this function in Δccm1 cells results, as it happens to any other unprocessed primary transcripts, in total degradation of 15S rRNA by mtEXO, whose mechanism of action is discussed.

The bRPS6-Family Protein RFC3 Prevents Interference by the Splicing Factor CFM3b during Plastid rRNA Biogenesis in Arabidopsis thaliana.

Plastid ribosome biogenesis is important for plant growth and development. REGULATOR OF FATTY ACID COMPOSITION3 (RFC3) is a member of the bacterial ribosomal protein S6 family and is important for lateral root development. rfc3-2 dramatically reduces the plastid rRNA level and produces lateral roots that lack stem cells. In this study, we isolated a suppressor of rfc three2 (sprt2) mutant that enabled recovery of most rfc3 mutant phenotypes, including abnormal primary and lateral root development and reduced plastid rRNA level. Northern blotting showed that immature and mature plastid rRNA levels were reduced, with the exception of an early 23S rRNA intermediate, in rfc3-2 mutants. These changes were recovered in rfc3-2 sprt2-1 mutants, but a second defect in the processing of 16S rRNA appeared in this line. The results suggest that rfc3 mutants may be defective in at least two steps of plastid rRNA processing, one of which is specifically affected by the sprt2-1 mutation. sprt2-1 mutants had a mutation in CRM FAMILY MEMBER 3b (CFM3b), which encodes a plastid-localized splicing factor. A bimolecular fluorescence complementation (BiFC) assay suggested that RFC3 and SPRT2/CFM3b interact with each other in plastids. These results suggest that RFC3 suppresses the nonspecific action of SPRT2/CFM3b and improves the accuracy of plastid rRNA processing.

Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy.

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

The 5.8S pre-rRNA maturation factor, M-phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis.

ObjectivesM‐phase phosphoprotein 6 (MPP6) is important for 5.8S pre‐rRNA maturation in somatic cells and was screened as a female fertility factor. However, whether MPP6 functions in oocyte meiosis and fertility is not yet known. We aimed to address this.Materials and MethodsMouse oocytes with surrounded nucleus (SN) or non‐surrounded nucleus (NSN) were used for all experiments. Peptide nanoparticle‐mediated antibody transfection was used to deplete MPP6. Immunofluorescence staining, immunohistochemistry and live tracker staining were used to examine MPP6 localization and characterize phenotypes after control or MPP6 depletion. High‐fidelity PCR and fluorescence in situ hybridization (FISH) were used to examine the localization and level of 5.8S rRNAs. Western blot was used to examine the protein level. MPP6‐EGFP mRNA microinjection was used to do the rescue.ResultsMPP6 was enriched within ovaries and oocytes. MPP6 depletion significantly impeded oocyte meiosis. MPP6 depletion increased 5.8S pre‐rRNA. The mRNA levels of MPP6 and 5.8S rRNA decreased within ageing oocytes, and MPP6 mRNA injection partially increased 5.8S rRNA maturation and improved oocyte quality.ConclusionsMPP6 is required for 5.8S rRNA maturation, meiosis and quality control in mouse oocytes, and MPP6 level might be a marker for oocyte quality.

A versatile cis-acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea.

General molecular principles of ribosome biogenesis have been well explored in bacteria and eukaryotes. Collectively, these studies have revealed important functional differences and few similarities between these processes. Phylogenetic studies suggest that the information processing machineries from archaea and eukaryotes are evolutionary more closely related than their bacterial counterparts. These observations raise the question of how ribosome synthesis in archaea may proceed in vivo. In this study, we describe a versatile plasmid-based cis-acting reporter system allowing to analyze in vivo the consequences of ribosomal RNA mutations in the model archaeon Haloferax volcanii. Applying this system, we provide evidence that the bulge-helix-bulge motif enclosed within the ribosomal RNA processing stems is required for the formation of archaeal-specific circular-pre-rRNA intermediates and mature rRNAs. In addition, we have collected evidences suggesting functional coordination of the early steps of ribosome synthesis in H. volcanii. Together our investigation describes a versatile platform allowing to generate and functionally analyze the fate of diverse rRNA variants, thereby paving the way to better understand the cis-acting molecular determinants necessary for archaeal ribosome synthesis, maturation, stability and function.

Modeling and plasmid expression of gene fragment of Ki67 protein biomarker

In eukaryotes, ribosome assembly is a rate-limiting step in ribosomal biogenesis that takes place in a distinctive subnuclear organelle, the nucleolus. How ribosomes get assembled at the nucleolar site by forming initial preribosomal complexes remains poorly characterized. In this study, using several human and murine cell lines, we developed a method for isolation of native mammalian preribosomal complexes by lysing cell nuclei through mild sonication. A sucrose gradient fractionation of the nuclear lysate resolved several ribonucleoprotein (RNP) complexes containing rRNAs and ribosomal proteins. Characterization of the RNP complexes with MS-based protein identification and Northern blotting–based rRNA detection approaches identified two types of preribosomes we named here as intermediate preribosomes (IPRibs) and composed preribosome (CPRib). IPRib complexes comprised large preribosomes (105S to 125S in size) containing the rRNA modification factors and premature rRNAs. We further observed that a distinctive CPRib complex consists of an 85S preribosome assembled with mature rRNAs and a ribosomal biogenesis factor, Ly1 antibody–reactive (LYAR), that does not associate with premature rRNAs and rRNA modification factors. rRNA-labeling experiments uncovered that IPRib assembly precedes CPRib complex formation. We also found that formation of the preribosomal complexes is nutrient-dependent because the abundances of IPRib and CPRib decreased substantially when cells were either deprived of amino acids or exposed to an mTOR kinase inhibitor. These findings indicate that preribosomes form via dynamic and nutrient-dependent processing events and progress from an intermediate to a composed state during ribosome maturation.