A 5 S rRNA-like secondary structure in the 7 SL RNA may define a ribosomal binding site of the signal recognition particle

A new secondary structure model for parts of the 7 SL RNA is proposed which indicates for a stretch of at least 40 bases a strong structural homology to the ribosomal protein L5 binding site of eukaryotic 5 S rRNA. It is suggested that the 5 S rRNA-like structural part of 7 SL RNA mediates binding of the signal recognition particle near to the peptidyl transferase center of the ribosome.     

Structure and evolution of the 7SL RNA component of the signal recognition particle

We have cloned and characterized cDNA copies of larval and adult Drosophila 7SL RNA. The Drosophila 7SL sequence shares 66.3% homology with that of human 7SL RNA. The homology is not evenly distributed along the sequence, but is concentrated in blocks in the central part of the molecule. We have analysed the secondary structure of Drosophila and human 7SL RNA free in solution by digestion with single and double strand specific nucleases. Similar experiments with the 7SL RNA bound to proteins within the signal recognition particle show essentially the same digestion pattern. A model of the secondary structure common to Drosophila and human 7SL RNA is presented.     

Low resolution three-dimensional models of the 7SL RNA of the signal recognition particle, based on an intramolecular cross-link introduced by mild irradiation with ultraviolet light

A number of intramolecular RNA-RNA cross-links were introduced into the human 7SL RNA by mild irradiation of a reconstituted signal recognition particle with ultraviolet light. Synthesis of radioactively labeled RNA was initiated in vitro from the T7 promoter. Smaller cross-linked complexes were generated by digestion of the RNA with RNase H in the presence of complementary deoxyoligonucleotides. Cross-linked complexes were separated from noncross-linked fragments by two-dimensional polyacrylamide gel electrophoresis. A close proximity between the stemloop around position 200 and the nucleotides at positions 67 to 76 was revealed by the absence of a characteristic oligonucleotide from the fingerprint of one of the complexes, suggesting a close spatial neighborhood between these two regions of the RNA. This and previous results, which described two different conformers of the RNA, were used to deduce two preliminary three-dimensional structure models of the 7SL RNA. The models differ in the base pairing scheme of the conserved core of the 7SL RNA.     

Induced structural changes of 7SL RNA during the assembly of human signal recognition particle

The eukaryotic signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein particle that targets secretory and membrane proteins to the endoplasmic reticulum. The binding of SRP54 to the S domain of 7SL RNA is highly dependent on SRP19. Here we present the crystal structure of a human SRP ternary complex consisting of SRP19, the M domain of SRP54 and the S domain of 7SL RNA. Upon binding of the M domain of SRP54 to the 7SL RNA-SRP19 complex, the asymmetric loop of helix 8 in 7SL RNA collapses. The bases of the four nucleotides in the long strand of the asymmetric loop continuously stack and interact with the M domain, whereas the two adenines in the short strand flip out and form two A-minor motifs with helix 6. This stabilizing interaction is only possible when helix 6 has been positioned parallel to helix 8 by the prior binding of SRP19 to the tetraloops of helices 6 and 8. Hence, the crystal structure of the ternary complex suggests why SRP19 is necessary for the stable binding of SRP54 to the S domain RNA.     

Evolution of secondary structure in the family of 7SL-like RNAs

Primate and rodent genomes are populated with hundreds of thousands copies of Alu and B1 elements dispersed by retroposition, i.e., by genomic reintegration of their reverse transcribed RNAs. These, as well as primate BC200 and rodent 4.5S RNAs, are ancestrally related to the terminal portions of 7SL RNA sequence. The secondary structure of 7SL RNA (an integral component of the signal recognition particle) is conserved from prokaryotes to distant eukaryotic species. Yet only in primates and rodents did this molecule give rise to retroposing Alu and B1 RNAs and to apparently functional BC200 and 4.5S RNAs. To understand this transition and the underlying molecular events, we examined, by comparative analysis, the evolution of RNA structure in this family of molecules derived from 7SL RNA.RNA sequences of different simian (mostly human) and prosimian Alu subfamilies as well as rodent B1 repeats were derived from their genomic consensus sequences taken from the literature and our unpublished results (prosimian and New World Monkey). RNA secondary structures were determined by enzymatic studies (new data on 4.5S RNA are presented) and/or energy minimization analyses followed by phylogenetic comparison. Although, with the exception of 4.5S RNA, all 7SL-derived RNA species maintain the cruciform structure of their progenitor, the details of 7SL RNA folding domains are modified to a different extent in various RNA groups. Novel motifs found in retropositionally active RNAs are conserved among Alu and B1 subfamilies in different genomes. In RNAs that do not proliferate by retroposition these motifs are modified further. This indicates structural adaptation of 7SL-like RNA molecules to novel functions, presumably mediated by specific interactions with proteins; these functions were either useful for the host or served the selfish propagation of RNA templates within the host genome.Abbreviations FAM fossil Alu element - FLAM free left Alu monomer - FRAM free right Alu monomer - L-Alu left Alu subunit - R-Alu right Alu subunit Correspondence to: D. LabudaDedicated to Dr. Robert Cedergren on the occasion of his 25th anniversary at the University of Montreal     

Structure-function analysis of the trypanosomatid spliced leader RNA.

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA.     

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.      

The 7S RNA from tomato leaf tissue resembles a signal recognition particle RNA and exhibits a remarkable sequence complementarity to viroids.

From tomato leaf tissue we sequenced and characterized a 7S RNA which consists of 299 nucleotides with either two or three additional uridine nucleotides at its 3'-terminus. About 56% of the nucleotides of this higher plant 7S RNA are in nearly identical positions as those of the human 7SL RNA which is an integral component of the signal recognition particle (SRP) that mediates protein translocation. Computer modelling and digestion studies with nucleases led to a secondary structure model for tomato 7S RNA, the overall shape of which is very similar to that of the human 7SL (SRP) RNA. This structural similarity strongly suggests that tomato 7S RNA is actually an SRP RNA and an integral part of the plant SRP, and that the protein translocation system of higher plants is very similar to the one operating in mammalian cells. Tomato SRP RNA contains a stretch of 36-53 nucleotides which exhibit a high degree of sequence complementarity to five viroid 'species' that cause disease in tomato. In the case of potato spindle tuber viroid and citrus exocortis viroid this complementarity spans the lower strand of the region, the nucleotides of which are known to modulate virulence. This extensive sequence complementarity could lead to a thermodynamically favoured base-pairing in vivo which renders the tomato SRP RNA a possible host target with which viroids could interact and thus incite disease.     

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.     

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.     

Diversity of 7 SL RNA from the signal recognition particle of maize endosperm.

An 11 S ribonucleoprotein particle was isolated from maize endosperm and shown to be functionally and structurally equivalent to the mammalian signal recognition particle. However, unlike animal cells which apparently contain a single 7 SL RNA species, maize endosperm contains a heterogeneous population of 7 SL RNA. To investigate this diversity, we have cloned and sequenced a number of the maize endosperm 7 SL RNAs isolated from functionally active SRP preparations. Some maize 7 SL RNAs are strikingly similar, differing by single base changes or short deletions; surprisingly, others share less than 70 percent sequence homology. Despite differences in primary sequence, nearly identical secondary structures can be suggested for all maize 7 SL RNAs, consistent with a proposed functional role in protein translocation for each of these RNAs. The amount of new available sequence data enabled us to define two conserved regions of presumed functional importance: A conserved sequence -G-N-A-R- in the center of a variable region which forms a well defined stem-loop and possibly is involved in an interaction with the 19 kDa protein of the SRP. Secondly, three short nucleotide stretches located in the central domain of 7 SL RNA may form part of a dynamic RNA-switch structure.     

A trinucleotide repeat-associated increase in the level of Alu RNA-binding protein occurred during the same period as the major Alu amplification that accompanied anthropoid evolution.

Nearly 1 million Alu elements in human DNA were inserted by an RNA-mediated retroposition-amplification process that clearly decelerated about 30 million years ago. Since then, Alu sequences have proliferated at a lower rate, including within the human genome, in which Alu mobility continues to generate genetic variability. Initially derived from 7SL RNA of the signal recognition particle (SRP), Alu became a dominant retroposon while retaining secondary structures found in 7SL RNA. We previously identified a human Alu RNA-binding protein as a homolog of the 14-kDa Alu-specific protein of SRP and have shown that its expression is associated with accumulation of 3'-processed Alu RNA. Here, we show that in early anthropoids, the gene encoding SRP14 Alu RNA-binding protein was duplicated and that SRP14-homologous sequences currently reside on different human chromosomes. In anthropoids, the active SRP14 gene acquired a GCA trinucleotide repeat in its 3'-coding region that produces SRP14 polypeptides with extended C-terminal tails. A C-->G substitution in this region converted the mouse sequence CCA GCA to GCA GCA in prosimians, which presumably predisposed this locus to GCA expansion in anthropoids and provides a model for other triplet expansions. Moreover, the presence of the trinucleotide repeat in SRP14 DNA and the corresponding C-terminal tail in SRP14 are associated with a significant increase in SRP14 polypeptide and Alu RNA-binding activity. These genetic events occurred during the period in which an acceleration in Alu retroposition was followed by a sharp deceleration, suggesting that Alu repeats coevolved with C-terminal variants of SRP14 in higher primates.     

The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence.

The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking. Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high abundance of methionine residues (M-domain). Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities. Upon proteolysis the intact G-domain was released from SRP, whereas the M-domain remained attached to the core of the particle. Reconstitution experiments demonstrated that the isolated M-domain associates with 7SL RNA in the presence of SRP19. In addition, we observed a specific binding of the M-domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA. This shows that the M-domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA. Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M-domain of SRP54. These results imply that the M-domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G-domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

The organization of the 7SL RNA in the signal recognition particle.

Digestion of the signal recognition particle (SRP) of dog pancreas with micrococcal nuclease results in the stepwise cleavage of the 300 nucleotide 7SL RNA moiety producing five major fragments approximately 220 (1), 150 (2), 72 (3), 62 (4) and 45 (5) nucleotides long. The RNA molecule is initially cut once yielding fragments 1 and 3. Further degradation releases fragments 2, 4 and 5. The introduction of the first nick into the 7SL RNA does not alter the structure nor the function of the SRP. Further degradation of the RNA results in disruption and loss of activity of the particle. The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme. The five major products of nuclease digestion together span almost the entire length of the 7SL RNA. Nicking occurs mainly around the boundary region between the central S sequence and the flanking Alu sequences constituting the 7SL RNA (1). The S fragment is bound to the four largest polypeptides while the 5' and 3' Alu fragments are associated with the two smallest protein constituents of the SRP.