End Maturation Research Articles

An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination

Polyadenylation is a co-transcriptional process that modifies mRNA 3′-ends in eukaryotes. In yeast, CF IA and CPF constitute the core 3′-end maturation complex. CF IA comprises Rna14p, Rna15p, Pcf11p and Clp1p. CF IA interacts with the C-terminal domain of RNA Pol II largest subunit via Pcf11p which links pre-mRNA 3′-end processing to transcription termination. Here, we analysed the role of Clp1p in 3′ processing. Clp1p binds ATP and interacts in CF IA with Pcf11p only. Depletion of Clp1p abolishes transcription termination. Moreover, we found that association of mutations in the ATP-binding domain and in the distant Pcf11p-binding region impair 3′-end processing. Strikingly, these mutations prevent not only Clp1p-Pcf11p interaction but also association of Pcf11p with Rna14p-Rna15p. ChIP experiments showed that Rna15p cross-linking to the 3′-end of a protein-coding gene is perturbed by these mutations whereas Pcf11p is only partially affected. Our study reveals an essential role of Clp1p in CF IA organization. We postulate that Clp1p transmits conformational changes to RNA Pol II through Pcf11p to couple transcription termination and 3′-end processing. These rearrangements likely rely on the correct orientation of ATP within Clp1p.

A novel function of Tis11b/BRF1 as a regulator ofDll4mRNA 3′-end processing

Tis11b/BRF1 belongs to the tristetraprolin family, the members of which are involved in AU-rich-dependent regulation of mRNA stability/degradation. Mouse inactivation of the Tis11b gene has revealed disorganization of the vascular network and up-regulation of the proangiogenic factor VEGF. However, the VEGF deregulation alone cannot explain the phenotype of Tis11b knockouts. Therefore we investigated the role of Tis11b in expression of Dll4, another angiogenic gene for which haploinsufficiency is lethal. In this paper, we show that Tis11b silencing in endothelial cells leads to up-regulation of Dll4 protein and mRNA expressions, indicating that Dll4 is a physiological target of Tis11b. Tis11b protein binds to endogenous Dll4 mRNA, and represses mRNA expression without affecting its stability. In the Dll4 mRNA 3' untranslated region, we identified one particular AUUUA motif embedded in a weak noncanonical polyadenylation (poly(A)) signal as the major Tis11b-binding site. Moreover, we observed that inhibition of Tis11b expression changes the ratio between mRNAs that are cleaved or read through at the poly(A) signal position, suggesting that Tis11b can interfere with mRNA cleavage and poly(A) efficiency. Last, we report that this Tis11b-mediated mechanism is used by endothelial cells under hypoxia for controlling Dll4 mRNA levels. This work constitutes the first description of a new function for Tis11b in mammalian cell mRNA 3'-end maturation.

The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer.

Ribonuclease P (RNase P) catalyzes the metal-dependent 5' end maturation of precursor tRNAs (pre-tRNAs). In Bacteria, RNase P is composed of a catalytic RNA (PRNA) and a protein subunit (P protein) necessary for function in vivo. The P protein enhances pre-tRNA affinity, selectivity, and cleavage efficiency, as well as modulates the cation requirement for RNase P function. Bacterial P proteins share little sequence conservation although the protein structures are homologous. Here we combine site-directed mutagenesis, affinity measurements, and single turnover kinetics to demonstrate that two residues (R60 and R62) in the most highly conserved region of the P protein, the RNR motif (R60-R68 in Bacillus subtilis), stabilize PRNA complexes with both P protein (PRNA•P protein) and pre-tRNA (PRNA•P protein•pre-tRNA). Additionally, these data indicate that the RNR motif enhances a metal-stabilized conformational change in RNase P that accompanies substrate binding and is essential for efficient catalysis. Stabilization of this conformational change contributes to both the decreased metal requirement and the enhanced substrate recognition of the RNase P holoenzyme, illuminating the role of the most highly conserved region of P protein in the RNase P reaction pathway.

Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3′‐end maturation and intron degradation

Polynucleotide phosphorylase (PNPase) catalyzes RNA polymerization and 3'→5' phosphorolysis in vitro, but its roles in plant organelles are poorly understood. Here, we have used in vivo and in vitro mutagenesis to study Arabidopsis chloroplast PNPase (cpPNPase). In mutants lacking cpPNPase activity, unusual RNA patterns were broadly observed, implicating cpPNPase in rRNA and mRNA 3'-end maturation, and RNA degradation. Intron-containing fragments also accumulated in mutants, and cpPNPase appears to be required for a degradation step following endonucleolytic cleavage of the excised lariat. Analysis of poly(A) tails, which destabilize chloroplast RNAs, indicated that PNPase and a poly(A) polymerase share the polymerization role in wild-type plants. We also studied two lines carrying mutations in the first PNPase core domain, which does not harbor the catalytic site. These mutants had gene-dependent and intermediate RNA phenotypes, suggesting that reduced enzyme activity differentially affects chloroplast transcripts. The interpretations of in vivo results were confirmed by in vitro analysis of recombinant enzymes, and showed that the first core domain affects overall catalytic activity. In summary, cpPNPase has a major role in maturing mRNA and rRNA 3'-ends, but also participates in RNA degradation through exonucleolytic digestion and polyadenylation. These functions depend absolutely on the catalytic site within the second duplicated RNase PH domain, and appear to be modulated by the first RNase PH domain.

Localization of Human RNase Z Isoforms: Dual Nuclear/Mitochondrial Targeting of the ELAC2 Gene Product by Alternative Translation Initiation

RNase Z is an endonuclease responsible for the removal of 3′ extensions from tRNA precursors, an essential step in tRNA biogenesis. Human cells contain a long form (RNase ZL) encoded by ELAC2, and a short form (RNase ZS; ELAC1). We studied their subcellular localization by expression of proteins fused to green fluorescent protein. RNase ZS was found in the cytosol, whereas RNase ZL localized to the nucleus and mitochondria. We show that alternative translation initiation is responsible for the dual targeting of RNase ZL. Due to the unfavorable context of the first AUG of ELAC2, translation apparently also starts from the second AUG, whereby the mitochondrial targeting sequence is lost and the protein is instead routed to the nucleus. Our data suggest that RNase ZL is the enzyme involved in both, nuclear and mitochondrial tRNA 3′ end maturation.

The fission yeast Schizosaccharomyces pombe has two distinct tRNase ZLs encoded by two different genes and differentially targeted to the nucleus and mitochondria

tRNase Z is the endonuclease that is involved in tRNA 3'-end maturation by removal of the 3'-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3'-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.

Structural basis for RNA trimming by RNase T in stable RNA 3′-end maturation

RNA maturation relies on various exonucleases to remove nucleotides successively from the 5' or 3' end of nucleic acids. However, little is known regarding the molecular basis for substrate and cleavage preference of exonucleases. Our biochemical and structural analyses on RNase T-DNA complexes show that the RNase T dimer has an ideal architecture for binding a duplex with a short 3' overhang to produce a digestion product of a duplex with a 2-nucleotide (nt) or 1-nt 3' overhang, depending on the composition of the last base pair in the duplex. A 'C-filter' in RNase T screens out the nucleic acids with 3'-terminal cytosines for hydrolysis by inducing a disruptive conformational change at the active site. Our results reveal the general principles and the working mechanism for the final trimming step made by RNase T in the maturation of stable RNA and pave the way for the understanding of other DEDD family exonucleases.

Impairment of mitochondrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia

We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNAIle. The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient's healthy mother. Functional characterization of the mutation revealed a six-fold reduced rate of tRNAIle precursor 3′ end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNAIle displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNAIle precursor 3′ end processing and contribute to the molecular pathomechanism of this mutation.

The C-terminal Region of the Exosome-associated Protein Rrp47 Is Specifically Required for Box C/D Small Nucleolar RNA 3′-Maturation

Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3′-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.

5′-end surveillance by Xrn2 acts as a shared mechanism for mammalian pre-rRNA maturation and decay

Ribosome biogenesis requires multiple nuclease activities to process pre-rRNA transcripts into mature rRNA species and eliminate defective products of transcription and processing. We find that in mammalian cells, the 5′ exonuclease Xrn2 plays a major role in both maturation of rRNA and degradation of a variety of discarded pre-rRNA species. Precursors of 5.8S and 28S rRNAs containing 5′ extensions accumulate in mouse cells after siRNA-mediated knockdown of Xrn2, indicating similarity in the 5′-end maturation mechanisms between mammals and yeast. Strikingly, degradation of many aberrant pre-rRNA species, attributed mainly to 3′ exonucleases in yeast studies, occurs 5′ to 3′ in mammalian cells and is mediated by Xrn2. Furthermore, depletion of Xrn2 reveals pre-rRNAs derived by cleavage events that deviate from the main processing pathway. We propose that probing of pre-rRNA maturation intermediates by exonucleases serves the dual function of generating mature rRNAs and suppressing suboptimal processing paths during ribosome assembly.

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.

Mammalian DEAD Box Protein Ddx51 Acts in 3′ End Maturation of 28S rRNA by Promoting the Release of U8 snoRNA

Biogenesis of eukaryotic ribosomes requires a number of RNA helicases that drive molecular rearrangements at various points of the assembly pathway. While many ribosome synthesis factors are conserved among all eukaryotes, certain features of ribosome maturation, such as U8 snoRNA-assisted processing of the 5.8S and 28S rRNA precursors, are observed only in metazoan cells. Here, we identify the mammalian DEAD box helicase family member Ddx51 as a novel ribosome synthesis factor and an interacting partner of the nucleolar GTP-binding protein Nog1. Unlike any previously studied yeast helicases, Ddx51 is required for the formation of the 3' end of 28S rRNA. Ddx51 binds to pre-60S subunit complexes and promotes displacement of U8 snoRNA from pre-rRNA, which is necessary for the removal of the 3' external transcribed spacer from 28S rRNA and productive downstream processing. These data demonstrate the emergence of a novel factor that facilitates a pre-rRNA processing event specific for higher eukaryotes.

A Divalent Cation Stabilizes the Active Conformation of the B. subtilis RNase P·Pre-tRNA Complex: A Role for an Inner-Sphere Metal Ion in RNase P

Metal ions interact with RNA to enhance folding, stabilize structure, and, in some cases, facilitate catalysis. Assigning functional roles to specifically bound metal ions presents a major challenge in analyzing the catalytic mechanisms of ribozymes. Bacillus subtilis ribonuclease P (RNase P), composed of a catalytically active RNA subunit (PRNA) and a small protein subunit (P protein), catalyzes the 5′-end maturation of precursor tRNAs (pre-tRNAs). Inner-sphere coordination of divalent metal ions to PRNA is essential for catalytic activity but not for the formation of the RNase P·pre-tRNA (enzyme–substrate, ES) complex. Previous studies have demonstrated that this ES complex undergoes an essential conformational change (to the ES ⁎ conformer) before the cleavage step. Here, we show that the ES ⁎ conformer is stabilized by a high-affinity divalent cation capable of inner-sphere coordination, such as Ca(II) or Mg(II). Additionally, a second, lower-affinity Mg(II) activates cleavage catalyzed by RNase P. Structural changes that occur upon binding Ca(II) to the ES complex were determined by time-resolved Förster resonance energy transfer measurements of the distances between donor–acceptor fluorophores introduced at specific locations on the P protein and pre-tRNA 5′ leader. These data demonstrate that the 5′ leader of pre-tRNA moves 4 to 6 Å closer to the PRNA·P protein interface during the ES-to-ES ⁎ transition and suggest that the metal-dependent conformational change reorganizes the bound substrate in the active site to form a catalytically competent ES ⁎ complex.

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.

Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA inSaccharomyces cerevisiae

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein-protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 3'-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed, one in transcription termination and one in 3'-end maturation. Impairment of the Sen1p-Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p-Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p-Rpb1p and Sen1p-Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression.

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.

Rrp17p Is a Eukaryotic Exonuclease Required for 5′ End Processing of Pre-60S Ribosomal RNA

SummaryRibosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 3′ end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 5′ end maturation. Here, we identify Rrp17p as a previously unidentified 5′–3′ exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 3′ end formation. Rrp17p is required for efficient exonuclease digestion of the mature 5′ ends of 5.8SS and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.

FLASH, a Proapoptotic Protein Involved in Activation of Caspase-8, Is Essential for 3′ End Processing of Histone Pre-mRNAs

3' end processing of histone pre-mRNA requires U7 snRNP, which binds downstream of the cleavage site and recruits the endonuclease CPSF-73. U7 snRNP contains a unique Sm ring in which the canonical SmD2 protein is replaced by Lsm11. We used the yeast two-hybrid system to identify binding partners of Lsm11 and selected the proapoptotic protein FLASH. Human FLASH interacts with Lsm11 in vitro and stimulates 3' end processing of histone pre-mRNA in mammalian nuclear extracts. We also identified the FLASH ortholog in Drosophila and demonstrate that it interacts with Lsm11 in vitro and in vivo. Drosophila FLASH localizes to histone locus bodies, and its depletion from fly cells inhibits U7-dependent processing, resulting in polyadenylation of histone mRNAs. These results demonstrate that FLASH is an essential factor required for 3' end maturation of histone mRNAs in both vertebrates and invertebrates and suggest a potential link between this process and apoptosis.

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5-insertion in the RNase J-encoding gene was compared to the wild-type and a difference in the length of the 5.8S-like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′-end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.

Trypanosoma bruceiSpliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2'-O-ribose methylation of the first four nucleotides and pseudouridine (psi) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2'-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL psi(28) formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3'-end maturation, and psi(28) formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.