The nucleoplasm and cytoplasm of eukaryotic cells contain a large number of small ribonucleoprotein (RNP) complexes, which are called small nuclear RNPs (snRNPs) and small cytoplasmic RNPs (scRNPs), respectively. They are composed of one or more proteins and a small stable RNA molecule (chain length <~300 nucleotides). The high abundance and the evolutionary conservation of RNA sequences within these small RNPs indicate that they have an important biological role. While a large body of information is available on the function of snRNPs in regulating nuclear RNA processing (see RNA Splicing), relatively little is known about the function of most scRNPs. Nonetheless, their cellular localization suggests a role in regulation of the last step of gene expression—that is, translation or disposition of newly synthesized proteins [reviewed by Baserga and Steitz (1)].
The discovery of the scRNPs was facilitated by autoantibodies present in sera of patients with autoimmune diseases in which antibodies against scRNPs components were found. The antibodies are usually targeted against a protein epitope, indicating that a common protein is associated with scRNPs of the same class. For example, antibodies against the 60-kDa Ro protein, which is a component of several scRNPs, also called Ro RNPs, were found in sera of patients with systemic lupus erythematosus (SLE) or with Sjogren’s syndrome (2-6). Sera of such patients frequently contain autoantibodies against the 50-kDa La protein (2, 3). These antigens were termed Ro and La, based on the names of the patients in whom they were first identified (3).
1. Ro RNPs
The Ro RNPs were first identified with the aid of anti-Ro autoantibodies (6). They contain one molecule of small cytoplasmic RNA that belongs to a family called Y RNAs, to indicate their cYtoplasmic location, and are assembled with the 60-kDa Ro protein and the 50-kDa La protein. The Y RNAs sequences, as well as the sequences of the Ro and La proteins, have been conserved during evolution. This, together with their high abundance (~105 copies per cell), suggests an important physiological role. However, their biological function is yet unknown [reviewed by Baserga and Steitz (1) and by Van Venrooij et al. (7)].
The Y RNAs are produced by transcription by RNA polymerase III. Four different Y RNA molecules, ranging in size between 69 to 112 nucleotides, were identified in humans and were termed Y1, Y3, Y4, and Y5 RNAs (Y2 is a truncated form of Y1). In different species, however, different numbers of types of Y RNAs were found associated in Ro RNPs. Thus, while humans and Xenopus cells contain all four types of Y RNAs, two types are found in mouse cells, and only one type is found in Caenorhabditis elegans (8-12).
The predicted secondary structure of sequenced Y RNAs can be drawn as a long base-paired stem composed of the 5′ and the 3′ ends of the molecule and an internal loop containing a pyrimidine-rich sequence (9, 13). Within the stem region, a highly conserved based-paired sequence with a bulged cytidine was proposed to be the binding site of the 60-kDa Ro protein (14, 15). The La antigen was proposed to bind to the 3′ oligo-uridylate sequence (15-17).
In Xenopus oocytes, the 60-kDa Ro protein is found also complexed with a heterogeneous population of 5S ribosomal RNA precursors that contain internal mutations. This binding is attributed to an alternative fold of the RNA in the mutant 5S rRNA. Because the mutant 5S rRNA precursors are processed inefficiently and are finally degraded, it was proposed that the Ro protein may function in a quality-control pathway of 5S rRNA biosynthesis (18, 19).
The 60-kDa Ro protein is an abundant and conserved protein that contains an RNA-recognition motif (RRM) (see RNA-Binding Proteins) of ~80 amino acid residues (10, 20). An additional protein of 52 kDa (Ro52), which is recognized by autoantibodies from patients with SLE and Sjogren’s syndrome, has been proposed to be a component of Ro RNPs in human cells (21), but other reports indicate otherwise (22, 23). The 52-kDa protein does not bind directly to the Y RNA, but probably associates with the Ro RNP through protein-protein interactions (24).
The 50-kDa La autoantigen is a highly abundant phosphoprotein that is found complexed with a variety of small RNAs to form small RNP particles [reviewed by Van Venrooij et al. (7)]. These include precursors to 5S rRNA, transfer RNA, 7S RNA, and Ro cytoplasmic Y RNAs, which are transcribed by RNA polymerase III (8, 25, 26). The La protein has an RNA recognition motif (RRM) (27). Its binding to the above-mentioned small RNAs occur via this RRM and is directed, at least in part, to a short uridylate sequence at the 3′ end of the relevant RNA (15-17).
The La protein has been proposed to be involved in a number of physiological processes within the cell’s nucleus. For example, experiments in vitro have indicated that the La protein is required as a transcription termination factor for RNA polymerase III transcripts (28-30), which is consistent with the protein’s ATPase activity that can melt DNA/RNA hybrids in vitro (31). Nevertheless, its association with scRNPs suggests a cytoplasmic physiological function as well. This view is supported by immunofluorescence experiments showing that the La protein is localized in the cytoplasm at sites of translation (32).
2. Viral scRNPs
An example of viral scRNPs are complexes of VAI and VAII small adenovirus RNAs that are transcribed by RNA polymerase III and have the La autoantigen as a common protein partner. These viral scRNPs are highly abundant in infected cells (~108 copies per cell) and are found in the cytoplasm. The ~160-nucleotide VAI RNA plays a role in enabling protein biosynthesis late in the adenovirus life cycle (33). Due to its high abundance and its binding to the interferon-induced 68-kDa kinase, it competes effectively with the cellular components that are involved in the interferon-induced transcription shutdown of translation (34, 35).
3. Signal Recognition Particle (SRP)
Another class of scRNPs are the signal recognition particles (SRPs) that help translocation of secretory proteins during translation. Mammalian SRPs are composed of 7SL RNA of ~300 nucleotides in length, which is complexed with six proteins having molecular weights of 9, 14, 19, 54, 68, and 72 kDa, to form an 11 S RNP particle [reviewed by Walter and Johnson (36) and by Lutcke (37)].
The 7SL SRP RNA is an abundant transcript of RNA polymerase III ( ~106 copies per cell). It is a member of a class of mammalian intermediate-repeat sequences called short interspersed elements ( SINES). The 7SL RNA has a remarkable sequence homology with the Alu family of repetitive sequences in primates and rodents. The "Alu domain" of the SRP consists of the Alu homologous region of 7SL RNA to which SRP9 and SRP14 are bound.
SRPs are required for cotranslational targeting of nascent secretory proteins to the endoplasmic reticulum. Translation of secretory proteins begins on ribosomes that are free in the cytosol. The SRP binds to the hydrophobic amino-terminal nascent peptide (15 to 30 amino acid residues) of the secreted nascent protein while it emerges from the large subunit of the ribosome. The binding arrests or delays the translation of the nascent polypeptide chain. The SRP then targets the nascent polypeptide-ribosome complex to the SRP receptor in the rough endoplasmic reticulum. The release of SRP from the signal sequence/ribosome complex requires GTP hydrolysis [reviewed by Walter and Johnson (36) by Lutcke (37), and by Bovia and Strub (38)]. Specifically, the 54-kDa protein, also called SRP54, binds directly to the signal peptide through a methionine-rich carboxyl-terminal domain. This protein is a GTP-binding protein, and when bound to the 7SL RNA it helps target the nascent chain by interacting with the heterodimeric SRP receptor. The function of the other proteins has not yet been characterized in detail. SRP19 aids the binding of SRP54 to the 7SL RNA, and the heterodimer SRP68/72 was proposed to interact with the a subunit of the SRP receptor. SRP68/72 and the Alu domain of SRP are required to confer elongation arrest activity of the particles.
The components of the SRP and SRP receptor were highly conserved during evolution. This is in accordance with their function in targeting proteins to the endoplasmic reticulum, which is shared by all eukaryotic organisms examined [reviewed by Wolin (39)].
4. Alu-Related scRNPs
Several Alu-related small RNAs were found to accumulate stably in the cytoplasm of rodent and primate cells. They are less abundant (103 to 10 4 copies per cell) and less well characterized than the above described scRNPs. Among them are the primate scAlu RNA (120 nucleotides), the rodent scB1 (140 nucleotides), and the BC200 RNA (200 nucleotides). The latter is expressed specifically in nerve cells by RNA polymerase III. These scRNAs have Alu-like sequences at their 5′ and 3′ ends, similar to the 7SL RNA, and can be drawn in a cruciform secondary structure. These Alu-like scRNAs are found complexed in small cytoplasmic RNPs and have been shown to bind the SRP14/19 heterodimer in vitro. Although the role of these scRNPs is yet unknown, a role in translation has been suggested [reviewed by Bovia and Strub (38)].
