Small ribonucleoproteins in Schizosaccharomyces pombe and Yarrowia lipolytica homologous to signal recognition particle.

Abstract

We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP). In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S. pombe and 270 nucleotides in Y. lipolytica) present in postribosomal salt extracts of the cytoplasm. The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S. pombe and Y. lipolytica, respectively. Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase HI promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded. Both of the yeast RNAs bind under stringent conditions to canine SRP proteins. Most importantly, RNase protection of the S. pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements ofthe mammalian and yeast RNA. Taken together these data suggest strongly that we have identified yeast SRP homologues.Fractionation of in vitro systems that mimic the translocation of nascent secretory proteins across the membrane of the mammalian endoplasmic reticulum (ER) has led to the identification ofa soluble ribonucleoprotein (RNP), the signal recognition particle (SRP), which is essential for targeting of nascent secretory proteins to the ER membrane. SRP is thought to function as an adapter between the cytoplasmic translation and the ER membrane-bound protein translocation machineries (1).SRP is a biochemically well-defined entity. It contains one RNA of 300 nucleotides (nt) (7SL RNA, here referred to as SRP RNA) and 6 polypeptides (2, 3). The canine SRP proteins can be separated from the RNA in native form and rebound to the RNA to reconstitute active SRP (4, 5). Studies on perturbed SRPs reconstituted either with modified SRP proteins or with only a subset of the proteins allowed us to dissect the functional contributions of individual SRP proteins (5,6). Heterologous reconstitutions of the canine proteins with Xenopus or Drosophila SRP RNAs also yield active particles in the in vitro assay, thus demonstrating the evolutionary conservation of SRP RNA (4). Phylogenetic analysis of the sequences from the human, amphibian, and insect RNAs has defined the secondary structure (7,8). The binding sites of two of the proteins (p19, a monomeric protein, and p68/72, a heterodimer) have been mapped (9) onto this structure.As a first step toward elucidating SRP function in vivo in genetically tractable organisms we have identified homologues of SRP in the yeasts Schizosaccharomyces pombe and Yarrowia lipolytica. Our strategy involved fractionation of yeast extracts to enrich for RNPs of similar b...

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA.

“…In contrast to the wide discrepancy in SRP54p binding to the two UUCG tetraloop mutants with inverted closing base pairs, the substitution of C-G for G-C in the context of the GAAA tetraloop has little effect on SRP54p binding. The fact that both UUCG replacement mutants exhibit conditional growth defects may indicate that subtle differences in structure from the wild-type GAAA loop reduce the binding efficiency of another factor, possibly SRP19p, which has been shown to protect residues in this region from RNase cleavage (16,23). …”

“…Model for interactions between SRP54p and SRP19p during assembly of SRP. The sequence of events depicted is based on the data presented here, together with earlier results from several other laboratories (7,16,23,24,32).…”

“…Evidence for cooperation between the two proteins during SRP assembly is provided by the observation that overexpression of Saccharomyces cerevisiae SRP54p suppresses a temperature-sensitive allele of the SEC65 gene, which encodes a putative SRP19p homolog in S. cerevisiae (7,24). In RNase footprinting experiments, the domain IV tetranucleotide loop, as well as the 5' side of the stem and preceding bulge, is protected from nuclease cleavage by canine SRP19 protein in both mammalian and fission yeast SRP RNAs (16,23). Although the pattern of protection did not change upon addition of SRP54p (23), in view of the 4.5S data, this finding likely reflects the limited digestion of the RNA by a-sarcin, the RNase used in these studies, rather than a lack of contact between the protein and domain IV.…”

“…Mammalian SRP is an 11S particle composed of a 300-nucleotide RNA (7SL) and six polypeptides (28,29). Previous work in several laboratories led to the discovery of a ribonucleoprotein in the fission yeast Schizosaccharomyces pombe that is similar in size to mammalian SRP and contains a 254-nucleotide homolog of human SRP RNA (3,16,18). Disruption of the srp7 gene demonstrated that this RNA is essential for viability in S. pombe (3,18).…”

Crystal structure of a signal recognition particle Alu domain in the elongation arrest conformation

“…1F), which we hypothesized might stabilize the closed conformation of the archaeal Alu domain. A similar structure is also predicted to exist in certain eubacteria which possess an Alu domain, such as Bacillus subtilis (Poritz et al 1988). Here, we describe the crystal structure of a chimeric Alu domain comprising the P. horikoshii Alu RNA (PhAlu) in complex with human SRP9/14.…”