Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.
We have developed a procedure for the affinity purification of small nuclear ribonucleoproteins (snRNPs) of Trypanosoma brucei (U2 and U4/U6 snRNPs), which are essential for trans splicing. Each of these snRNPs can be specifically and efficiently selected from T. brucei extracts through biotinylated antisense 2'-0-methylated RNA oligonucleotides immobilized on streptavidin-agarose. Protein analysis revealed a set of five low molecular weight polypeptides common to the U2 and U4/U6 snRNPs and the spliced leader RNP. In addition, several U2 and U4/U6 snRNP-specific protein components were identified. Using monoclonal antibodies against human snRNP proteins, we could not detect any significant cross-reaction with the trypanosomal U2 snRNP proteins. Thus, the trypanosomal snRNPs exhibit principal differences from the higher eukaryotic snRNPs not only in their RNA but also in their protein components.trans splicing in trypanosomatids and nematodes follows a two-step mechanism of cleavage-ligation reactions analogous to nuclear pre-mRNA splicing (cis splicing) of other eukaryotes. In trypanosomes, a mini-exon derived from the 5' end of the spliced leader (SL) RNA is trans-spliced onto every protein-coding exon of long, polycistronic pre-mRNAs (reviewed in refs. 1 and 2). As in cis splicing, small nuclear RNAs (snRNAs) function as essential splicing factors in trans splicing (3): trypanosomal U2, U4, and U6 RNAs have been identified (4-6) that are in the form of U2, U4/U6, and U6 small nuclear ribonucleoproteins (snRNPs) (7, 27); however, no U5 RNA homologue is known in trypanosomes. The SL RNP can be considered a chimeric molecule that possesses characteristics of an snRNP and carries the mini-exon sequence at the SL RNA 5' terminus, thereby possibly performing the role of the U1 snRNP (8-10). In contrast to the mammalian snRNPs (reviewed in ref. 11), we know little about the RNA-protein structure of the trypanosomal snRNPs and their mechanism of action during trans splicing. The Trypanosoma brucei snRNAs deviate in many aspects from their strongly conserved eukaryotic counterparts (reviewed in ref.2). Thus, through the characterization and comparison of cis-and trans-spliceosomal snRNPs, we expect to gain more information about the specific requirements of trans splicing. We have developed procedures for the affinity purification of SL, U2, and U4/U6 snRNPs on the basis of biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotides, resulting in the identification of a set of five common proteins and additional, snRNP-specific proteins. Interestingly, no immunological relationship of these proteins with mammalian snRNP proteins could be detected.
Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4͞U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.T rans splicing in trypanosomes is an essential step in the expression of all mRNAs and results in joining of a short, noncoding miniexon sequence [spliced leader (SL)] to each of the protein-coding sequences that are part of long polycistronic precursors (reviewed in ref. 1). As in the cis-spliceosome, small nuclear RNAs (snRNAs) U2, U4, and U6, in addition to the SL RNA, are essential cofactors for trans splicing (2). In addition, the trypanosomatid U5 snRNA has been identified (3-5). Surprisingly, Schnare and Gray (6) recently discovered also a U1-like small RNA in the trypanosomatid species Crithidia fasciculata and Leishmania tarentolae, which may be required for cis splicing of internal introns such as the one of the poly(A) polymerase gene (7).We had previously established affinity purification procedures that allowed the identification of protein components in the trans-spliceosomal small nuclear ribonucleoproteins (snRNPs) from Trypanosoma brucei. A set of at least five polypeptides of 8.5, 10, 12.5, 14, and 15 kDa, which we have called common proteins, was detected originally in the SL RNP, the U2 snRNP, and the U4͞U6 snRNP (8). Common proteins were localized by immunof luorescence predominantly in the nucleoplasm of trypanosomes (9). They make up a stable core shared between these snRNPs and bind to an snRNA region resembling the Sm sequence of cis-spliceosomal snRNPs (10). Using polyclonal Abs that we generated against a mixture of four of these proteins (8.5, 10, 12.5, and 14 kDa), we showed later that these core proteins are present in the U5 snRNP (4) and the SLA (spliced leader-associated) RNP (11). For the trypanosomal U4 and U5 snRNAs, we ...
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