Mature 18S rRNA Research Articles

The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits

RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases-hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells. Furthermore, we demonstrate that depletion of hRio1 by RNA interference affects the last step of 18S rRNA maturation and causes defects in the recycling of several trans-acting factors (hEnp1, hRio2, hLtv1, hDim2/PNO1, and hNob1) from pre-40S subunits in the cytoplasm. Although the effects of hRio1 and hRio2 depletion are similar, we show that the two kinases are not fully interchangeable. Moreover, rescue experiments with a kinase-dead mutant of hRio1 revealed that the kinase activity of hRio1 is essential for the recycling of the endonuclease hNob1 and its binding partner hDim2 from cytoplasmic pre-40S. Kinase-dead hRio1 is trapped on pre-40S particles containing hDim2 and hNob1 but devoid of hEnp1, hLtv1, and hRio2. These data reveal a role of hRio1 in the final stages of cytoplasmic pre-40S maturation.

Mycobacterium smegmatis RNase J is a 5′‐3′ exo‐/endoribonuclease and both RNase J and RNase E are involved in ribosomal RNA maturation

The presence of very different sets of enzymes, and in particular the presence of RNase E and RNase J, has been used to explain significant differences in RNA metabolism between the two model organisms Escherichia coli and Bacillus subtilis. However, these studies might have somewhat polarized our view of RNA metabolism. Here, we identified a RNase J in Mycobacterium smegmatis that has both 5'-3' exo- and endonucleolytic activity. This enzyme coexists with RNase E in this organism, a configuration that enabled us to study how these two key nucleases collaborate. We demonstrate that RNase E is responsible for the processing of the furA-katG transcript in M. smegmatis and that both RNase E and RNase J are involved in the 5' end processing of all ribosomal RNAs. In contrast to B. subtilis, the activity of RNase J, although required in vivo for 23S rRNA maturation, is not essential in M. smegmatis. We show that the pathways for ribosomal RNA maturation in M. smegmatis are quite different from those observed in E. coli and in B. subtilis. Studying organisms containing different combinations of key ribonucleases can thus significantly broaden our view of the possible strategies that exist to direct RNA metabolism.

The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3′ end of 16S rRNA

Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides ( ) near the 3' end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the to double mutation severely affects the viability of bacteria. However, whether Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.

RNase G participates in processing of the 5′-end of 23S ribosomal RNA

In Escherichia coli, primary rRNA transcripts must be processed by a complex process in which several ribonucleases are involved in order to generate mature 16S, 23S, and 5S rRNA molecules. While it is known that RNase G, a single-stranded RNA-specific endoribonuclease encoded by the rng gene, plays an active role in the maturation of the 5'-end of 16S rRNA, its involvement in the maturation of the 5'-end of 23S rRNA remains unclear. Here we show that E. coli cells deleted for the rng gene accumulate the 23S rRNA precursor containing an extra 77 nucleotides at its mature 5'-end. In vitro cleavage assays show that RNase G cleaves synthetic RNA containing a sequence encompassing the 5'-end to 77 nucleotides upstream of mature 23S rRNA at two sites present in single-stranded regions. Our results suggest the involvement of RNase G in the processing of the 5'-region of 23S rRNA precursors.

Downregulation of NIN/RPN12 binding protein inhibit the growth of human hepatocellular carcinoma cells

The ribosome assembly factor NIN/RPN12 binding protein (Nob1) has been suggested to be essential for processing of the 20S pre-rRNA to the mature 18S rRNA, and is also reported to participate in proteasome biogenesis. However, it is unclear whether Nob1 is involved in tumor cells growth. The aim of this study was to determine whether the suppression of Nob1 by short hairpin RNA (shRNA) inhibits the growth of human hepatocellular carcinoma (HCC) cells. Recombinant lentiviral shRNA expression vector carrying Nob1 was constructed and then infected into human HCC cell line SMMC-7721. The growth properties of SMMC-7721/pGCSIL-GFP-shNC and pGCSIL-GFP-shNob1 cells were determined by MTT, BrdU incorporation assay, and flow cytometric analysis. In addition, the colony formation and tumor growth ability in nude mice were detected to define the function of Nob1 in cell transformation and tumorigenesis. Our data showed that the growth and proliferation of SMMC-7721/pGCSIL-GFP-shNob1 cells were significantly reduced compared with the SMMC-7721/pGCSIL-GFP-shNC. In addition, the colony formation was impaired after the suppression of Nob1 in SMMC-7721 cells. And in vivo, the tumor formation ability of the SMMC-7721/pGCSIL-GFP-shNob1 cells was significantly reduced compared with the control cells. Our data support that Nob1 is an important regulator of the tumorigenic properties of human HCC and could be used as a candidate therapeutic target in human HCC.

Roles of the 5′‐phosphate sensor domain in RNase E

Viable mutations affecting the 5'-phosphate sensor of RNase E, including R169Q or T170A, become lethal when combined with deletions removing part of the non-catalytic C-terminal domain of RNase E. The phosphate sensor is required for efficient autoregulation of RNase E synthesis as RNase E R169Q is strongly overexpressed with accumulation of proteolytic fragments. In addition, mutation of the phosphate sensor stabilizes the rpsT P1 mRNA as much as sixfold and slows the maturation of 16S rRNA. In contrast, the decay of other model mRNAs and the processing of several tRNA precursors are unaffected by mutations in the phosphate sensor. Our data point to the existence of overlapping mechanisms of substrate recognition by RNase E, which lead to a hierarchy of efficiencies with which its RNA targets are attacked.

Suppression of a cold‐sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway.

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+² dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+² (translationally inactive) and high Mg+² (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

The late stages of 30S and 50S ribosomal subunits biogenesis have been studied in a wild-type (wt) strain of Escherichia coli (MC4100) subjected to a severe heat stress (45–46°C). The 32S and 45S ribosomal particles (precursors to 50S subunits) and 21S ribosomal particles (precursors to 30S subunits) accumulate under these conditions. They are authentic precursors, not degraded or dead-end particles. The 21S particles are shown, by way of a modified 3′5′ RACE procedure, to contain 16S rRNA unprocessed, or processed at its 5′ end, and not at the 3′ end. This implies that maturation of 16S rRNA is ordered and starts at its 5′-terminus, and that the 3′-terminus is trimmed at a later step. This observation is not limited to heat stress conditions, but it also can be verified in bacteria growing at a normal temperature (30°C), supporting the idea that this is the general pathway. Assembly defects at very high temperature are partially compensated by plasmid-driven overexpression of the DnaK/DnaJ chaperones. The ribosome assembly pattern in wt bacteria under a severe heat stress is therefore reminiscent of that observed at lower temperatures in E. coli mutants lacking the chaperones DnaK or DnaJ.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A₀, A₁, and A₂. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature-sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.

Las1L Is a Nucleolar Protein Required for Cell Proliferation and Ribosome Biogenesis

Ribosome biogenesis is a highly regulated process ensuring that cell growth (increase in biomass) is coordinated with cell proliferation. The formation of eukaryotic ribosomes is a multistep process initiated by the transcription and processing of rRNA in the nucleolus. Concomitant with this, several preribosomal particles, which transiently associate with numerous nonribosomal factors before mature 60S and 40S subunits are formed and exported in the cytoplasm, are generated. Here we identify Las1L as a previously uncharacterized nucleolar protein required for ribosome biogenesis. Depletion of Las1L causes inhibition of cell proliferation characterized by a G1 arrest dependent on the tumor suppressor p53. Moreover, we demonstrate that Las1L is crucial for ribosome biogenesis and that depletion of Las1L leads to inhibition of rRNA processing and failure to synthesize the mature 28S rRNA. Taken together, our data demonstrate that Las1L is essential for cell proliferation and biogenesis of the 60S ribosomal subunit.

Utp25p, a nucleolar Saccharomyces cerevisiae protein, interacts with U3 snoRNP subunits and affects processing of the 35S pre‐rRNA

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. Two intermediate complexes, pre-40S and pre-60S, are formed at the early stages of 35S pre-rRNA processing and give rise to the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, some ribosomal proteins and processing factors. The novel yeast protein Utp25p has previously been identified in the nucleolus, an indication that this protein could be involved in ribosome biogenesis. Here we show that Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p, and affects 18S rRNA maturation. Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels. Our results indicate that Utp25p is a novel SSU processome subunit involved in pre-40S maturation.

A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis

Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.

An Archaeal Dim2-Like Protein, aDim2p, Forms a Ternary Complex with a/eIF2α and the 3′ End Fragment of 16S rRNA

Dim2p is a eukaryal small ribosomal subunit RNA processing factor required for the maturation of 18S rRNA. Here we show that an archaeal homolog of Dim2p, aDim2p, forms a ternary complex with the archaeal homolog of eIF2α, a/eIF2α, and the RNA fragment that possesses the 3′ end sequence of 16S rRNA both in solution and in crystal. The 2.8-Å crystal structure of the ternary complex reveals that two KH domains of aDim2p, KH-1 and -2, are involved in binding the anti-Shine–Dalgarno core sequence (CCUCC-3′) and a highly conserved adjacent sequence (5′-GGAUCA), respectively, of the target rRNA fragment. The surface plasmon resonance results show that the interaction of aDim2p with the target rRNA fragment is very strong, with a dissociation constant of 9.8 × 10 − 10 M, and that aDim2p has a strong nucleotide sequence preference for the 3′ end sequence of 16S rRNA. On the other hand, aDim2p interacts with the isolated α subunit and the intact αβγ complex of a/eIF2, irrespective of the RNA binding. These results suggest that aDim2p is a possible archaeal pre-rRNA processing factor recognizing the 3′ end sequence (5′-GAUCACCUCC-3′) of 16S rRNA and may have multiple biological roles in vivo by interacting with other proteins such as a/eIF2 and aRio2p.

Evolutionarily Conserved Function of RRP36 in Early Cleavages of the Pre-rRNA and Production of the 40S Ribosomal Subunit

Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.

Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity

Ribosomal protein S5 is critical for small ribosomal subunit (SSU) assembly and is indispensable for SSU function. Previously, we identified a point mutation in S5, (G28D) that alters both SSU formation and translational fidelity in vivo, which is unprecedented for other characterized S5 mutations. Surprisingly, additional copies of an extraribosomal assembly factor, RimJ, rescued all the phenotypes associated with S5(G28D), including fidelity defects, suggesting that the effect of RimJ on rescuing the miscoding of S5(G28D) is indirect. To understand the underlying mechanism, we focused on the biogenesis cascade and observed defects in processing of precursor 16S (p16S) rRNA in the S5(G28D) strain, which were rescued by RimJ. Analyses of p16S rRNA-containing ribosomes from other strains further supported a correspondence between the extent of 5(') end maturation of 16S rRNA and translational miscoding. Chemical probing of mutant ribosomes with additional leader sequences at the 5(') end of 16S rRNA compared to WT ribosomes revealed structural differences in the region of helix 1. Thus, the presence of additional nucleotides at the 5(') end of 16S rRNA could alter fidelity by changing the architecture of 16S rRNA in translating ribosomes and suggests that fidelity is governed by accuracy and completeness of the SSU biogenesis cascade.

Coordinated Regulation of 23S rRNA Maturation in Escherichia coli

In Escherichia coli, rRNAs are transcribed as precursors and require processing at the 3' and 5' ends to generate mature RNA molecules. The largest of these RNAs, 23S rRNA, is matured at the 3' end by a set of exonucleases and at the 5' end by an unknown RNase. Whether the 3' and 5' maturation steps occur independently or are coupled has previously been unclear. By assessing the levels of precursors accumulating at the 3' and 5' ends, we provide evidence that these processes may be linked. Thus, each of several conditions that led to precursor accumulation at one end also did so at the other end. We also observed that each end undergoes maturation at similar rates, suggesting that the two processes could be coupled. Finally, we provide evidence that processing at the 3' end facilitates 5'-end maturation. A model to explain the basis for the observed directionality of the reactions is proposed. This information will aid in the search for the enzyme responsible for final maturation of the 5' end of 23S rRNA.

Analysis of ribosomal RNA transcription termination and 3′ end processing in Leishmania amazonensis

The control of gene expression in the human parasite Leishmania occurs mainly at the post-transcriptional level. Nevertheless, basic cell processes such as ribosome biogenesis seem to be conserved. Mature ribosomal RNAs (rRNAs) are synthesized from typical RNA polymerase I (Pol I) promoters and processed by pathways analogous to other eukaryotes. To further understand Pol I transcription control in these parasites, we have analyzed transcription termination and processing of the rDNA in Leishmania amazonensis. 3′-end S1 mapping of rRNA precursors identified three termini, one corresponding to the mature 28S rRNA and two to the rDNA intergenic spacer (IGS), termed T1 and T2, for precursors which are 185 and 576 nucleotides longer, respectively. Both T1 and T2 are associated with conserved G + C rich elements that have the potential to form hairpin structures and T-rich clusters. We found that two fragments of 423 bp and 233 bp, flanking sites T1 and T2 respectively when placed upstream of the green fluorescent protein gene ( GFP), negatively affected the Pol I-driven transcription of this gene, which suggests the presence of a transcription terminator element in these regions. Deletion analysis pointed to a CCCTTTT heptamer as part of the putative terminator and suggested that the hairpins are processing signals.

Maturation of 23S rRNA inBacillus subtilisin the Absence of Mini-III

23S rRNA maturation in Bacillus subtilis is catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5'-to-3' exoribonuclease RNase J1 and the 3'-to-5' exoribonucleases, principally RNase PH and YhaM.