Signal recognition particle components in the nucleolus

Abstract

The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and͞or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves. T he nucleolus long has been known as a dense subnuclear structure at which ribosomal genes are clustered and ribosomal transcription, rRNA processing, and ribosomal subunit assembly occurs (1). More recently, it has become clear that the RNA components of other ribonucleoproteins also may localize to the nucleolus (2, 3), including the signal recognition particle (SRP) (4). Protein components of both RNase P and RNase MRP also recently have been shown to be localized in the nucleolus (5). The hypothesis has been advanced that the nucleolus not only has evolved to carry out ribosome synthesis but, in fact, is the site of assembly of and͞or interaction between multiple ribonucleoprotein machines, many of which are involved in protein synthesis (6). However, no information regarding the potential nucleolar localization of the other components of the SRP has been available.The SRP is a ribonucleoprotein that arrests translation of secretory or membrane proteins and docks the nascent polypeptide-ribosome complex at receptors on the endoplasmic reticulum (ER), whereupon translation is resumed to direct the protein into the membrane assembly or secretory pathways (7-9). In mammalian cells, the SRP contains six proteins (7) and an Ϸ300-nt RNA (10) that previously had been known as 7S RNA or 7SL RNA (11-13). In an early study it was found that ...

Control of box C/D snoRNP assembly by N 6 ‐methylation of adenine

“…Searching more widely we noted that the human signal recognition particle (SRP) RNA Alu domain contains a sequence that had been considered as a putative k‐turn, with the normal consecutive G·A and A·G base pairs (Fig 7). Moreover, like box C/D snoRNP, the SRP is assembled in the nucleolus 55, 56. This has a HS 2 HS 1 structure 57, but the known Haloarcula marismortui k‐turn Kt‐58 has the same secondary structure and a closely similar sequence.…”

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

“…The assembly pathway of the Alu RNP in vivo is likely to be the same as in vitro but must be seen in the context of the complete SRP and in the context of the compartmentalization of the eukaryotic cell, which leads to a spatial separation of events+ Combining our results on the assembly of the Alu domain with previously published data and hypotheses, we suggest the following scenario for the assembly of SRP in vivo+ SRP9 and SRP14 first heterodimerize in the cytoplasm and subsequently enter the nucleoplasm+ There SRP9/14 binds cotranscriptionally to the Alu RNA 59 domain enhancing transcription in cis (Emde et al+, 1997)+ After termination of transcription, the Alu RNA 39 domain bends into the closed conformation and exposes the 39 end for nuclear processing (Chen et al+, 1998)+ Then SRP19 and SRP68/72 join the S-domain RNA in the nucleolus before the subparticle gets exported to the cytoplasm where SRP54 adds on in a final step (Politz et al+, 2000)+ There is experimental evidence that Alu RNA contains all necessary elements for efficient nuclear export (Jacobson & Pederson, 1998)+ Although the presence of SRP9/14 in the nucleus and its cotranscriptional association with Alu RNA have not explicitly been demonstrated, we consider the scenario to be quite plausible+ Recent data from the distantly related yeast (y)SRP (Ciufo & Brown,FIGURE 7. Hierarchical assembly pathway of the SRP Alu domain+ Heterodimerization of SRP9 (red) and SRP14 (green) is a prerequisite for Alu RNA 59 domain (blue) binding+ This interaction induces the RNA 39 domain (cyan) to flip around and bind across the interface of the 59 domain complex+ The model is consistent with the RNase V1 probing data (light green and light red phosphate-ribose backbone) and explains the role of SRP9 K41 (violet) in 39 domain binding (contacted oxygens in yellow)+ At very high particle concentrations, an intermolecular 59 domain-39 domain interaction could be favored, leading to particle dimerization+ 2000) show that ySRP14 can be detected in the nucleolus where it takes part in the assembly of a core particle together with ySRP21, ySRP68, ySRP72, and ySRP RNA (scR1)+ The model presented above is equally likely to be valid for the assembly, transport, and function of non-SRP Alu RNPs+…”

“…The assembly of the S-domain of SRP has been found to be cooperative in vitro (Walter & Blobel, 1983) and to partially take place in the nucleolus and in the cytoplasm in vivo (Politz et al+, 2000)+ This underlines the notion that the assembly of multicomponent particles like SRP or the ribosome in vivo has to be imagined in a stepwise, hierarchical, and controlled order with precise temporal and spatial separation of events+ Concerning the assembly and function of the Alu domain of SRP it has been shown previously that SRP9/14 heterodimerization is a prerequisite for Alu RNA binding )+ We have also determined experimentally the minimal Alu RNA folding domain, SA86 (Weichenrieder et al+, 1997), which consists of a distinct 59 domain (nt 1-47 of human SRP RNA) covalently linked to a 39 domain (nt 48-64 of SRP RNA base paired to nt 282-300; Fig+ 1)+ Alu RNA constructs designed according to SA86 have been shown to be sufficient for accurate SRP RNA 39 end processing (Chen et al+, 1998) and for efficient export of SRP RNA to the cytoplasm (Jacobson & Pederson, 1998)+ A construct corresponding to the SA86 59 domain strongly activates transcription by RNA polymerase III (Emde et al+, 1997)+ Structures with very similar RNA and protein components to the SRP Alu domain are found in other, non-SRP contexts (Fig+ 1) and understanding the assembly and conformational state of these Alu RNPs will also be critical to understanding their function+ Most interesting are the neuron-specific BC200 RNP, which migrate into dendrites to possibly regulate localized protein translation (Kremerskothen et al+, 1998) and the Alu retroposition intermediates repeated in tandem, which are responsible for the creation of 10-12% of the human genome in the form of Alu elements (Mighell et al+, 1997)+ Alu retroposition is an ongoing process that must have had a significant impact on the evolution of the human genome (Kazazian, 1998 Weichenrieder et al+, 2000)+ In solution, the free SA50 is likely to be largely prefolded but with enhanced flexibility between the helical stacks to the extent that the tertiary interactions between the two loops (including three Watson-Crick base pairs) may be partially disrupted (Figs+ 2C, 4B)+ Specific binding of SRP9/14 to the conserved U-turn of the 59 domain rigidifies the structure and promotes formation of the tertiary interactions+ Such flexibility between helical RNA stacks is of general importance for the process of RNA folding and might be functionally relevant in many cases (Batey & Doudna, 1998)+ In summary, the preferential binding of SRP9/14 to the Alu RNA 59 domain is a novel observation and the structural rearrangements that occur in the RNA upon protein binding illustrate the role of proteins to assist large RNAs in adopting their physiologically relevant conformations+…”

Rrs1 Is Involved in Endoplasmic Reticulum Stress Response in Huntington Disease

“…In recent proteomic analysis of the nucleolus, molecules that were previously considered exclusively resident in the ER membrane were found in both compartments, such as the signal recognition particle 68 (SRP68) (28). The assembly of the presignal recognition particle actually seems to occur in the nucleolus before being targeted to the nuclear pores.…”