The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization

The human La autoantigen (hLa) protein is a predominantly nuclear phosphoprotein that contains three potential RNA binding domains referred to as the La motif and the RNA recognition motifs RRMs 1 and 2. With this report, we differentiated the contribution of its three RNA binding domains to RNA binding by combining in vitro and in vivo assays. Also, surface plasmon resonance technology was used to generate a model for the sequential contribution of the RNA binding domains to RNA binding. The results indicated that the La motif may contribute to specificity rather than affinity, whereas RRM1 is indispensable for association with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucial for the interaction with various RNAs in vivo, although needed for full-affinity binding in vitro. Moreover, earlier studies suggest that RNA binding by hLa may direct its subcellular localization. As shown previously for RRM1, deletion of RNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed after deletion of the La motif. Here we discuss a model for precursor RNA binding based on a sequential association process mediated by RRM1 and the La motif.     

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.     

Two neuronal,nuclear-localized RNA binding proteins involved in synaptic transmission

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.     

Analysis of RNA motifs

RNA motifs are directed and ordered stacked arrays of non-Watson-Crick base pairs forming distinctive foldings of the phosphodiester backbones of the interacting RNA strands. They correspond to the 'loops' - hairpin, internal and junction - that intersperse the Watson-Crick two-dimensional helices as seen in two-dimensional representations of RNA structure. RNA motifs mediate the specific interactions that induce the compact folding of complex RNAs. RNA motifs also constitute specific protein or ligand binding sites. A given motif is characterized by all the sequences that fold into essentially identical three-dimensional structures with the same ordered array of isosteric non-Watson-Crick base pairs. It is therefore crucial, when analyzing a three-dimensional RNA structure in order to identify and compare motifs, to first classify its non-Watson-Crick base pairs geometrically.     

Down regulation of extramacrochaetae mRNA by a Drosophila neural RNA binding protein Rbp9 which is homologous to human Hu proteins.

Rbp9 is an RNA binding protein expressed mainly in the central nervous system of adult Drosophilamelanogaster. Rbp9 shares a high degree of sequence similarity with human neural proteins referred to as Hu antigens. Hu antigens bind to U-rich mRNA destabilizing elements with a high affinity and, thus, have been implicated as regulators of mRNA stability. Using in vitro RNA binding assays, we found that Rbp9 binds strongly to poly U sequences. We then employed a Selex system to identify a consensus Rbp9 binding site (UUUXUUUU). Information obtained from the Selex results allowed the detection of two repeats of the Rbp9 consensus binding sequence in the 3' untranslated region of extramacrochaetae mRNA. UV crosslinking experiments demonstrated that Rbp9 interactsspecifically with emc mRNA. The requirement of Rbp9 protein in the down regulation of emc mRNA was confirmed by northern (RNA) analysis, which revealed that the level of emc mRNA increased 10-fold in rbp9 mutant flies. Taken together with the in vitro RNA binding results, the genetic evidence obtained strongly supports the hypothesis that Rbp9 functions as a regulator of RNA stability.     

RNA binding proteins in the RNA interference phenomenon

The ability of short RNAs (21-27 nucleotides) to silence genes containing homologous nucleotide sequences is related to RNA silencing. The pathways of short RNAs (siRNA and microRNA) biogenesis from their precursors, double stranded and hairpin RNAs respectively, are briefly reviewed. The functioning of specific RNA binding domains found for the first time in the proteins operating in RNA interference (RNAi) is considered. The interactions of these domains with the earlier well known RNA binding modules in RNAi proteins are described.     

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.     

The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding

Prp24 is an essential yeast U6 snRNP protein with four RNA recognition motifs (RRMs) that facilitates the association of U4 and U6 snRNPs during spliceosome assembly. Genetic interactions led to the proposal that RRMs 2 and 3 of Prp24 bind U6 RNA, while RRMs 1 and 4 bind U4 RNA. However, the function of each RRM has yet to be established through biochemical means. We compared the binding of recombinant full-length Prp24 and truncated forms lacking RRM 1 or RRM 4 with U6 RNA. Contrary to expectations, we found that the N-terminal segment containing RRM 1 is important for high-affinity binding to U6 RNA and for discrimination between wild-type U6 RNA and U6 with point mutations in the 3' intramolecular stem-loop. In contrast, deletion of RRM 4 and the C terminus did not significantly alter the affinity for U6 RNA, but resulted in the formation of higher order Prp24.U6 complexes. Truncation and internal deletion of U6 RNA mapped three Prp24-binding sites, with the central site providing most of the affinity for Prp24. A newly identified temperature-sensitive lethal point mutation in RRM 1 is exacerbated by mutations in the U6 RNA telestem, as is a mutation in RRM 2, but not one in RRM 3. We propose that RRMs 1 and 2 of yeast Prp24 bind the same central site in U6 RNA that is bound by the two RRMs of human Prp24, and that RRMs 3 and 4 bind lower affinity flanking sites, thereby restricting the stoichiometry of Prp24 binding.     

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ~7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.     

Phosphorylation of human Argonaute proteins affects small RNA binding

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5'-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5' phosphate of the small RNA.