Both catalytic steps of nuclear pre-mRNA splicing are reversible

Nuclear pre-messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions. The presence of introns in spliceosomal RNAs of certain fungi has suggested that splicing may be reversible; however, this has never been demonstrated experimentally. By using affinity-purified spliceosomes, we have shown that both catalytic steps of splicing can be efficiently reversed under appropriate conditions. These results provide considerable insight into the catalytic flexibility of the spliceosome.     

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.     

Argonaute2 is the catalytic engine of mammalian RNAi

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.     

A specific amino acid binding site composed of RNA

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

Model substrates for an RNA enzyme

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.     

Splicing of messenger RNA precursors

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.     

The design and catalytic properties of a simplified ribonuclease P RNA

Ribonuclease P (RNase P) RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from the 5' ends of pre-transfer RNAs in eubacteria. Phylogenetic variation according to recently proposed secondary structure models was used to identify structural elements of the RNase P RNA that are dispensable for catalysis. A simplified RNase P RNA that consists only of evolutionarily conserved features was designed, synthesized, and characterized. Although the simplified RNA (Min 1 RNA) is only 263 nucleotides in length, in contrast to the 354 to 417 nucleotides of naturally occurring RNase P RNAs, its specificity of pre-tRNA cleavage is identical to that of the native enzymes. Moreover, the catalytic efficiencies of the Min 1 RNA and the native RNA enzymes are similar. These results focus the search for the catalytic elements of RNase P RNAs to their conserved structure.     

Ribozyme-catalyzed transcription of an active ribozyme

A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template.     

A microRNA in a multiple-turnover RNAi enzyme complex

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.     

HSPC117 is the essential subunit of a human tRNA splicing ligase complex

Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.     

Association of TALS developmental disorder with defect in minor splicing component U4atac snRNA

The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.     

Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon

The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.