Reverse self-splicing of group II intron RNAs in vitro.

Group II introns, which are classed together on the basis of a conserved secondary structure, are found in organellar genes of lower eukaryotes and plants. Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats. A remarkable feature of group II introns is their self-splicing activity in vitro. In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation. By virtue of its ability to specifically bind the 5' exon, the intron can also catalyse such reactions on exogenous RNA substrates. This sequence-specific attachment could enable group II introns to integrate into unrelated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron. Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro. This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'-5' phosphodiester bond of the lariat RNA into the 3'-5' bond of the reconstituted 5' splice junction.     

Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction

The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.     

Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.     

Trans-spliced leader RNA exists as small nuclear ribonucleoprotein particles in Caenorhabditis elegans

Maturation of some messenger RNAs in the nematode Caenorhabditis elegans involves the acquisition of a 22-base leader at their 5' ends. This 22-base leader, called the spliced leader (SL), is derived from the 5' end of a precursor RNA of 90-100 bases, called spliced leader RNA (SL RNA). SL RNA is transcribed from a 1-kilobase DNA repeat which also encodes the 5S ribosomal RNA. A subset of mRNAs in C. elegans acquire SL from SL RNA by a trans-splicing mechanism. SL behaves as a 5' exon in the trans-splicing reaction. Using antisera against the Sm antigen that is associated with small nuclear ribonucleoprotein particles (snRNPs), we precipitated SL RNA from extracts of C. elegans, indicating that it is bound by the Sm antigen in vivo. SL RNA also possesses the unique trimethylguanosine (m32,2,7G) cap characteristic of most small nuclear RNAs. Therefore, SL RNA is a chimaeric molecule, made up of an snRNA attached to a 5' exon and is a constituent of a snRNP.     

Does Q beta replicase synthesize RNA in the absence of template?

Q beta replicase, in the absence of added template, will synthesize RNA autocatalytically. A variety of small RNa species, termed '6S RNAs' are generated. As this reaction purportedly occurs in the absence of template, it has been termed 'de novo' RNA synthesis. The question of whether Q beta replicase can polymerize replicatable RNA molecules, without instruction from a template, has important evolutionary implications. The finding that Q beta replicase was able to synthesize RNA de novo was based on (1) failure to find contaminating RNA in Q beta replicase preparations; (2) differences in the sizes of products of apparently identical reactions; and (3) kinetic differences between template-instructed and de novo reactions. Here wer describe a procedure for production of Q beta replicase lacking one of its subunits, ribosomal protein S1, involving column chromatography in the presence of a low concentration of urea. We show that the resulting highly purified enzyme will not synthesize detectable RNA in the absence of added template. We show also that the ability to perform a reaction kinetically indistinguishable from the de novo synthesis reaction can be restored to the highly purified enzyme by adding a heat-stable, alkali-labile component of Q beta replicase preparations. Thus our findings suggest that, in the novo reaction, Q beta replicase is replicating previously undetected contaminating RNA molecules.     

Why do nucleic acids have 3'5' phosphodiester bonds?

Details of the stereochemistry of the 2'5' and 3'5' dinucleoside monophosphates of polynucleotides have been delineated in aqueous solution using nuclear magnetic resonance spectroscopy. Incorporation of these experimentally determined geometries into the structure of polynucleotides reveals that the intrinsic spatial configurations of the 2'5' bonds cannot support helical structures whereas the geometries of 3'5' bonds allow the formation of helical configurations for RNA.     

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.     

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).     

Telomerase primer specificity and chromosome healing

Chromosome healing by de novo telomere addition at nontelomeric sites has been well characterized in several organisms. The Tetrahymena telomerase ribonucleoprotein uses an internal RNA template to catalyse d(TTGGGG)n telomere addition to the 3' end of telomeric sequence in vitro and in vivo. Studies of telomerase RNA indicated that hybridization of the RNA template region, 5'-CAACCCCAA-3', to the 3' end of single-stranded telomeric oligonucleotides might be important for primer recognition and utilization. The apparent requirement of telomerase for pre-existing telomeric sequence has raised questions regarding its role in chromosome healing. We report here that Tetrahymena telomerase can specifically elongate single-stranded DNA oligonucleotides whose termini are not complementary to the RNA template sequence 5'-CAACCCCAA-3'. These data suggest that telomerase may be able to heal chromosomes directly in vivo.     

The human RNA kinase hClp1 is active on 3' transfer RNA exons and short interfering RNAs

RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer, synthetic siRNAs display a 5' OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5' end. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing and messenger RNA 3'-end formation machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5' end of the 3' exon during human tRNA splicing, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3'-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.     

Dicer recognizes the 5' end of RNA for efficient and accurate processing

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3' end but also the 5' end, with the cleavage site determined mainly by the distance (～22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5' pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5'-pocket mutant. Thus, 5'-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.     

Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.