Characterization of human autoantibodies that selectively precipitate the 7SL RNA component of the signal recognition particle

The signal recognition particle (SRP), which consists of the 7SL RNA molecule associated with six polypeptides ranging between 9,000 and 72,000 m.w., mediates the translocation of newly synthesized proteins across the endoplasmic reticulum. We have characterized autoantibodies that are directed against this particle from two patients with rheumatic diseases. These sera immunoprecipitated the 7SL RNA from whole extracts of HeLa cells radiolabeled with 32P, but no RNA from deproteinized cell extracts. From 35S-methionine-labeled cell extracts, they immunoprecipitated a single polypeptide of 54,000 m.w. that is consistent with a known SRP component. Sucrose density gradient studies confirmed that this protein co-migrated with the 7SL RNA, indicating the likelihood that it is physically associated with this RNA. Thus, the 54,000 m.w. SRP protein, which is essential for the SRP functions of elongation arrest and translocation, appears to be a preferential target for human autoimmune responses. Human autoantibodies that recognize the SRP should be useful adjuncts to animal antisera for studies of the structure and function of this particle.     

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.     

Changes in 7SL RNA conformation during the signal recognition particle cycle.

The structure of 7SL RNA has been probed by chemical modification followed by primer extension, using four substrates: (i) naked 7SL RNA; (ii) free signal recognition particle (SRP); (iii) polysome bound SRP; and (iv) membrane bound SRP. Decreasing sensitivity to chemical modification between these different substrates suggests regions on 7SL RNA that: bind proteins associated with SRP might interact with ribosomes; and are protected by binding to membranes. Other areas increase in chemical sensitivity, exemplified by a tertiary interaction present in naked 7SL RNA but not in free SRP. Such changes suggest that 7SL RNA changes its conformation during the SRP cycle. These conformational changes could be a necessary component to move through the SRP cycle from one stage to the next.     

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.     

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.     

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.     

Structure,function and evolution of the signal recognition particle

The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)-SRP complex. The RNC-SRP complex then docks in a GTP-dependent manner with a membrane-anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.     

The trypanosomatid signal recognition particle consists of two RNA molecules,a 7SL RNA homologue and a novel tRNA-like molecule

Trypanosomatids are ancient eukaryotic parasites affecting humans and livestock. Here we report that the trypanosomatid signal recognition particle (SRP), unlike all other known SRPs in nature, contains, in addition to the 7SL RNA homologue, a short RNA molecule, termed sRNA-85. Using conventional chromatography, we discovered a small RNA molecule of 85 nucleotides co-migrating with the Leptomonas collosoma 7SL RNA. This RNA molecule was isolated, sequenced, and used to clone the corresponding gene. sRNA-85 was identified as a tRNA-like molecule that deviates from the canonical tRNA structure. The co-existence of these RNAs in a single complex was confirmed by affinity selection using an antisense oligonucleotide to sRNA-85. The two RNA molecules exist in a particle of approximately 14 S that binds transiently to ribosomes. Mutations were introduced in sRNA-85 that disrupted its putative potential to interact with 7SL RNA by base pairing; such mutants were unable to bind to 7SL RNA and to ribosomes and were aberrantly distributed within the cell. We postulate that sRNA-85 may functionally replace the truncated Alu domain of 7SL RNA. The discovery of sRNA-85 raises the intriguing possibility that sRNA-85 functional homologues may exist in other lower eukaryotes and eubacteria that lack the Alu domain.     

7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles

The virion incorporation of 7SL, the RNA component of the host signal recognition particle (SRP), has been shown for several simple retroviruses. Data here demonstrate that 7SL is also packaged by HIV-1, in sevenfold molar excess of genomic RNA. Viral determinants of HIV-1 genome and primer tRNA packaging were not required for 7SL incorporation, as virus-like particles with only minimal assembly components efficiently packaged 7SL. The majority of 7SL within cells resides in ribonucleoprotein complexes bound by SRP proteins, and most SRP protein exists in signal recognition particles. However, Western blot comparison of virion and cell samples revealed that there is at least 25-fold less SRP p54 protein per 7SL RNA in HIV-1 particles than in cells. Comparing 7SL:actin mRNA ratios in virions and cells revealed that 7SL RNA appears selectively enriched in virions.     

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.     

Prediction of signal recognition particle RNA genes

We describe a method for prediction of genes that encode the RNA component of the signal recognition particle (SRP). A heuristic search for the strongly conserved helix 8 motif of SRP RNA is combined with covariance models that are based on previously known SRP RNA sequences. By screening available genomic sequences we have identified a large number of novel SRP RNA genes and we can account for at least one gene in every genome that has been completely sequenced. Novel bacterial RNAs include that of Thermotoga maritima, which, unlike all other non-gram-positive eubacteria, is predicted to have an Alu domain. We have also found the RNAs of Lactococcus lactis and Staphylococcus to have an unusual UGAC tetraloop in helix 8 instead of the normal GNRA sequence. An investigation of yeast RNAs reveals conserved sequence elements of the Alu domain that aid in the analysis of these RNAs. Analysis of the human genome reveals only two likely genes, both on chromosome 14. Our method for SRP RNA gene prediction is the first convenient tool for this task and should be useful in genome annotation.     

Identification of chloroplast signal recognition particle RNA genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.     

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2- terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.     

Structure of SL4 RNA from the HIV-1 packaging signal.

The NMR-based structure is described for an RNA model of stem-loop 4 (SL4) from the HIV-1 major packaging domain. The GAGA tetraloop adopts a conformation similar to the classic GNRA form, although there are differences in the details. The type II tandem G.U pairs have a combination of wobble and bifurcated hydrogen bonds where the uracil 2-carbonyl oxygen is hydrogen-bonded to both G,H1 and G,H2. There is the likelihood of a Na(+) ion coordinated to the four carbonyl oxygens in the major groove for these G.U pairs and perhaps to the N7 lone pairs of the G bases as well. A continuous stack of five bases extends over nearly the whole length of the stem to the base of the loop in the RNA 16mer: C15/U14/G13/G5/C6. There is no evidence for a terminal G.A pair; instead, G1 appears quite unrestrained, and A16 stacks on both C15 and G2. Residues G2 through G5 exhibit broadened resonances, especially G3 and U4, suggesting enhanced mobility for the 5'-side of the stem. The structure shows G2/G3/U4 stacking along the same strand, nearly isolated from interaction with the other bases. This is probably an important factor in the signal broadening and apparent mobility of these residues and the low stability of the 16mer hairpin against thermal denaturation.     

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.