Biogenesis of the signal recognition particle - Semantic Scholar

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Biogenesis of the signal recognition particle Eileen Leung1 and Jeremy D. Brown2 RNA Biology Group and Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, U.K.

Abstract Assembly of ribonucleoprotein complexes is a facilitated quality-controlled process that typically includes modification to the RNA component from precursor to mature form. The SRP (signal recognition particle) is a cytosolic ribonucleoprotein that catalyses protein targeting to the endoplasmic reticulum. Assembly of SRP is largely nucleolar, and most of its protein components are required to generate a stable complex. A pre-SRP is exported from the nucleus to the cytoplasm where the final protein, Srp54p, is incorporated. Although this outline of the SRP assembly pathway has been determined, factors that facilitate this and/or function in quality control of the RNA are poorly understood. In the present paper, the SRP assembly pathway is summarized, and evidence for the involvement of both the Rex1p and nuclear exosome nucleases and the TRAMP (Trf4–Air2–Mtr4p polyadenylation) adenylase in quality control of SRP RNA is discussed. The RNA component of SRP is transcribed by RNA polymerase III, and both La, which binds all newly transcribed RNAs generated by this enzyme, and the nuclear Lsm complex are implicated in SRP RNA metabolism.

Ribonucleoprotein biogenesis Non-coding RNAs have a long (and ever-increasing) list of functions in cells. However, few, if any, of these RNAs act alone; instead, they are assembled into multi-component machines, RNPs (ribonucleoproteins). Many RNPs are catalytic, and these activities may lie in RNA or protein components. Some RNPs, and particularly the ribosome and spliceosome, are spectacularly complicated, comprising several RNA species and many proteins. Others, such as RISCs (RNA-induced silencing complexes), RNase P and telomerase are relatively simple, containing single RNAs and a few proteins. Biogenesis pathways of all RNPs are, however, similar in many respects, and most involve multiple steps and factors. RNA processing steps are intimately linked into the assembly of many RNPs, with endo- and/or exo-nucleases removing portions of the primary transcript to generate the mature RNA. Other trans-acting factors modify the RNA, facilitate RNA folding and protein binding and may impose quality control on the assembly process. A further common theme is that steps in RNP assembly frequently occur in different cellular compartments to where the mature complexes function, with transport factors moving complexes between compartments. Many factors that act in RNP assembly are involved in the biogenesis of multiple complexes. These include proteins that associate with the 3 end of RNAs, including the conserved RNA-binding protein La, the nuclear Lsm2–8, polyadenylating TRAMP (Trf4–Air2–Mtr4p polyadenylation) and nucleolytic exosome complexes. Endo- and exoKey words: exosome, La, nuclease, RNA processing, signal recognition particle (SRP), yeast. Abbreviations used: pol, RNA polymerase; RNP, ribonucleoprotein; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA; SRP, signal recognition particle; TRAMP, Trf4–Air2–Mtr4p polyadenylation. 1 Present address: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K. 2 To whom correspondence should be addressed (email [email protected]).

Biochem. Soc. Trans. (2010) 38, 1093–1098; doi:10.1042/BST0381093

nucleases also typically have multiple substrates. For example, targets of the yeast Saccharomyces cerevisiae RNAse III homologue Rnt1p include precursors of U3 and other box C/D snoRNAs (small nucleolar RNAs) as well as preribosomal RNA and pre-U1, U4 and U5 snRNAs (small nuclear RNAs). Regulating the activities of various factors that bind and modify pre-RNPs such that processing and assembly steps are correctly ordered is an important part of the biogenesis process. A key function of La is its association with newly synthesized pol (RNA polymerase) III transcripts, to which it binds through their 3 uridine [oligo(U)] tract [1,2]. La promotes the folding of at least some of the RNAs that it binds [3] and association with La protects RNAs from 3 →5 exonucleolytic digestion. Analysis of pre-tRNA maturation in yeast revealed that both the order and mechanism by which 5 - and 3 -extensions of many pre-tRNAs are removed is altered in cells lacking La: binding of La favours removal of the 3 -trailer by endonucleolytic cleavage, influencing the pathway of pre-tRNA maturation [4]. La also has roles in metabolism of intermediates in the processing of pol IItranscribed snRNAs that contain a uridine-rich 3 -terminus (examples include the U1–U5 snRNAs and U3 snoRNA) [5,6]. The Lsm2–8 complex stably associates with the U6 snRNP [7–9], and is bound to precursor forms of many other RNAs, including RNase P RNA, tRNAs, snoRNAs and rRNAs. Depletion of Lsm proteins leads to defects in processing of many RNAs, including pre-tRNAs and prerRNAs. Furthermore, the Lsm complex has been suggested to promote association of processing factors with their RNA substrates [10,11]. The nuclear TRAMP complex, through its Trf4p subunit, polyadenylates a variety of RNAs [12–14]. A second related complex contains an alternative adenylase subunit, Trf5p, which, although redundant with Trf4p for growth, targets a largely different set of RNAs [15,16]. TRAMP complexes  C The

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contribute to RNA surveillance in the nucleus/nucleolus, promoting processing and/or degradation of RNAs through their ability to recruit and stimulate activities of the nuclear exosome. The nuclear exosome has two associated nucleases: (i) the exonuclease Rrp6p, and (ii) Rrp44p, which has both exo- and endo-nucleolytic activities [17–22]. The addition of poly(A) tails to RNAs by TRAMP complexes is proposed to stimulate exosome activity, though turnover of many RNA substrates of Trf4p appears to take place with similar efficiency when its adenylase activity is compromised [16]. Activities of both Rrp6p and Rrp44p are reported to be stimulated by the TRAMP complex [23,24]. How La, Lsm proteins, TRAMP and exosome complexes and other factors interact with each other and their substrates are key issues in understanding RNA processing and RNP assembly. In the case of the exosome and perhaps other nucleases involved in the processing of precursor RNAs, a decision (or balance) also has to be struck between degradation and processing.

The SRP (signal recognition particle) The SRP is an abundant universally conserved RNP that catalyses the docking of ribosomes synthesizing pre-secretory and membrane proteins to the translocation apparatus found in the endoplasmic reticulum membrane in eukaryotes and the cell membrane of prokaryotes [25,26]. Newly synthesized pre-secretory proteins in eukaryotes carry Nterminal hydrophobic signal sequences, that are bound by SRP when they emerge from the ribosome. In eukaryotes, the binding of SRP to the signal sequence and ribosome leads to slowing of peptide synthesis, known as ‘elongation arrest’, a conserved function that facilitates the coupling of protein synthesis with the translocation process [27–29]. Elongation arrest is released when ribosomes dock on to the integral membrane proteins that comprise the translocation apparatus, a process that requires a specific SRP receptor. Although SRP function is analogous in all organisms, its composition varies somewhat. Mammalian SRP comprises a ∼300 nt RNA (7SL) and six polypeptides with masses of 9, 14, 19, 54, 68 and 72 kDa organized into two domains, namely the S-domain and Alu-domain [30]. The S-domain contains the central region of the RNA together with SRP19, SRP54, SRP68 and SRP72, of which SRP54 binds the signal sequences of pre-secretory proteins. The Alu-domain contains the 5 - and 3 -terminal regions of the RNA along with SRP9 and SRP14 and harbours the elongation arrest activity of the complex. Fungal SRP also consists of one RNA (termed scR1 in yeast) and six proteins, Srp14p, Srp21p (homologous with mammalian SRP9), Sec65p (homologous with mammalian SRP19), Srp54p, Srp68p and Srp72p [31,32]. In contrast, Alu-domain proteins are absent in trypanosome SRP, and the complex contains an additional tRNA-like RNA associated with the Alu-domain [33,34]. Bacterial and archaeal SRP complexes are simpler, having fewer protein subunits and, in general, smaller RNA components.  C The

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SRP assembly SRP assembly has been studied in both yeast and higher eukaryotes, and in both cases is largely nucleolar. This was first suggested from the observation that on injection of fluorescently labelled 7SL into the nucleus, it accumulated in the nucleolus before localizing to the cytoplasm [35]. The subcellular distribution of GFP (green fluorescent protein)tagged SRP proteins and endogenous SRP RNA was also consistent with this, several proteins and the RNA having nucleolar as well as cytoplasmic pools [36]. Most of the yeast SRP proteins (Srp72p, Srp68p, Srp21p and Srp14p) also localize to both the nucleolus and cytoplasm [37,38]. These proteins are required for assembly of a stable complex containing SRP RNA, and are termed the core SRP proteins [31]. The small amount of RNA remaining in cells lacking any one of the core proteins is found throughout the nucleus [37,38]. Sec65p is found throughout the nucleus and cytoplasm, whereas Srp54p is exclusively cytoplasmic. Import of the core SRP proteins into the nucleus requires the importins Pse1p and Kap123p/Yrb4p [37], which also import ribosomal proteins [39,40], and may constitute a nucleolar import pathway. These data led to a model in which SRP biogenesis commences within the nucleolus where a preSRP complex containing the four core proteins bound to the RNA is assembled. SRP19/Sec65p binds this complex, which is competent for export from the nucleus. Export itself requires the leucine-rich nuclear-export-sequence-binding protein Xpo1p/Crm1p and the nucleoporins Nsp1p and Nup159p [37,38]. The final step in SRP assembly is binding of Srp54p, which, in mammalian as well as yeast cells, localizes exclusively to the cytoplasm [35,37,38].

Quality control of SRP RNA Beyond transport adapters and nucleoporins that facilitate movement of SRP proteins and pre-SRP in and out of the nucleus, factors that function in SRP biogenesis have yet to be determined. Several are, however, implicated in SRP RNA metabolism.

La Like all nascent pol III transcripts, SRP RNA associates with La. However, unlike most other RNAs transcribed by this polymerase, SRP RNA retains its 3 oligo(U) tract in the mature RNP. Indeed, isolation of SRP from yeast cells under native conditions using affinity tags appended to protein components yields La as a co-purifying protein, suggesting that the 3 end of the RNA is available to bind La in the mature complex [41,42]. Interestingly, the majority of SRP RNA in mammalian cells is trimmed to leave a single U of the initial oligo(U) tract and monoadenylated modifications that would abrogate La binding [43]. Adenylation of SRP RNA is conserved, and is also seen in Xenopus and a small fraction of the yeast RNA [44]. The presence of La bound to SRP RNA could impede or prevent export of the complex from the nucleus. Adenylation might facilitate or be key in release of

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Figure 1 Rrp44p activity is necessary for correct localization of yeast SRP RNA A yeast strain containing the rrp44-1 thermosensitive mutation grown at 23◦ C or shifted to 36◦ C for 30 min was fixed, and SRP RNA and DNA were detected by FISH (fluorescence in situ hybridization), and images were captured and processed as described in [38]. Arrows highlight the accumulated SRP RNA adjacent to the nuclear DNA in the nucleolus.

the complex from the nucleus. A similar proportion of nuclear and cytoplasmic SRP RNA was found to be adenylated in Xenopus oocytes, and thus, if the RNA has to be adenylated to escape the nucleus, the modification must be reversible [44]. A candidate enzyme capable of adenylating SRP RNA was purified [45]. Also known as poly(A) polymerase γ , an orthologue of the standard poly(A) polymerase, this has both non-specific and CPSF (cleavage and polyadenylation specificity factor)/AAUAAA-dependent polyadenylation activity [46,47]. Specificity was lost during purification of the enzyme, and a recombinant version of it polyadenylated RNA non-specifically [45]. Whether or not this enzyme is responsible for monoadenylation of SRP RNA in vivo is then unclear.

Lsm proteins Lsm proteins may have a role in SRP RNA metabolism and a small fraction of yeast SRP RNA is associated with Lsm proteins [10]. A significant proportion (>30%) of SRP RNA can be isolated from yeast cells expressing a Protein Atagged La, but the amount is reduced ∼10-fold when either Lsm3 or Lsm5 are depleted [10]. Since the great majority of SRP RNA is cytoplasmic, whereas La localizes to the nucleus, the extensive association between La and SRP RNA

in this experiment probably occurred once cells were broken and Lsm proteins could facilitate association of La with the mature RNP. Whether or not the observation reflects a physiological role for the Lsm complex is unclear.

TRAMP, exosome and Rex1p Examination of yeast strains carrying mutations to its associated nucleases revealed that, although loss of Rrp6p did not affect SRP RNA localization, inactivation of a thermosensitive version of Rrp44p by incubation at the nonpermissive temperature, resulted in accumulation of SRP RNA in the nucleolus [37]. These results were obtained following extensive (4 h) incubation at the non-permissive temperature, and SRP RNA is also present in increased amounts at much shorter time points, suggesting a direct, rather than potentially indirect, effect (Figure 1). A small fraction of SRP RNA accumulates as a truncated species in yeast lacking the TRAMP adenylase Trf4p ([48] and E. Leung and J.D. Brown, unpublished work), indicating that TRAMP facilitates degradation of SRP RNA beyond a particular point. Consistent with a role for the TRAMP complex in SRP RNA turnover, microarray analysis indicates that the amount of the RNA is significantly increased in cells lacking Trf4p [16]. This is not, however, reflected on  C The

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Figure 2 Pathways for SRP RNA turnover and SRP assembly See the text for details. SRP RNA is shown as folded [50] at all stages in the pathway, but folding may be influenced by La, SRP proteins and other factors during the assembly process.

Northern blots, where SRP RNA levels appear similar, regardless of the presence or absence of Trf4p ([48] and E. Leung and J.D. Brown, unpublished work). The reason for this discrepancy is unclear. Further examination of SRP RNA levels in strains lacking TRAMP and exosome activities will hopefully uncover the effects that these mutations have on the RNA. Interestingly, nuclear accumulation of SRP RNA in cells in which a thermosensitive Rrp44p is inactivated is not accompanied by appearance of a truncated RNA species ([37] and E. Leung and J.D. Brown, unpublished work). However, depletion of Rrp44p does lead to accumulation of truncated SRP RNA, and Rrp44p is not required for steps leading to generation of this species (E. Leung and J.D. Brown, unpublished work). The separation of nuclear accumulation and degradation intermediate phenotypes indicates two functions for Rrp44p in SRP RNA metabolism. One is important for degradation of a fraction of the RNA discarded during the biogenesis process, whereas the other leads to correct localization of SRP RNA. A second nuclease implicated in turnover of SRP RNA is Rex1p, a 3 →5 exonuclease that contributes to the 3 -end trimming of a number of RNAs, and is required for the 3  C The

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end trimming of 5S rRNA [49]. Truncated SRP RNA is not seen when Rex1p is removed from trf4Δ cells, and thus Rex1p is important for steps in degradation that lead to generation of truncated SRP RNA. Copela et al. [48] also found that overexpression of La suppressed the truncated SRP RNA in cells lacking Trf4p. La can thus out-compete the machinery that would otherwise degrade a proportion of SRP RNA.

Model Cumulatively, the observations above suggest a turnover pathway for SRP RNA in which it is initially trimmed by Rex1p (Figure 2). TRAMP and Rrp44p act in later steps in turnover, being particularly important in accessing sequences beyond the point represented by the truncated SRP RNA that accumulates in Trf4p-deficient or Rrp44p-depleted cells. This pathway may remove transcripts that do not bind La initially (or cannot bind La), and may also remove SRP RNA that does not proceed efficiently through later steps, particularly assembly with core SRP proteins. Binding of core proteins to the RNA stabilizes it, and, since the pre-RNP can, at this stage, be exported from the nucleus [37,38], an export signal must be revealed in the complex at this stage. Whether

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export is mediated directly by Xpo1p or through adaptor(s) is unknown. Several core SRP proteins have potential nuclear export sequences, but, to date, strains expressing proteins with mutations to these have not revealed any deficit in localization of the complex to the cytoplasm (J.D. Brown and R.W. van Nues, unpublished work). Given the observed 3 adenylation of SRP RNA, could it be that this reflects association with TRAMP and exosome complexes as part of a pathway that both monitors integrity/folding of the RNA and acts to pass the RNA from initial binding factors to core proteins and or export factors?

Funding Work was supported by Research Career Development Fellowship from the Wellcome Trust [grant number G117/395] and Senior Non-Clinical Research Fellowship from the Medical Research Council [grant number 046300] and the Biotechnology and Biological Sciences Research Council [grant number BB/F014910/1] (to J.D.B.) and a Ph.D. studentship from the Biotechnology and Biological Sciences Research Council (to E.L.).

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