Sm proteins form stable ribonucleoprotein (RNP) complexes with small nuclear (sn)RNAs and are core components of the eukaryotic spliceosome. In vivo, the assembly of Sm proteins onto snRNAs requires the survival motor neurons (SMN) complex. Several reports have shown that SMN protein binds with high affinity to symmetric dimethylarginine (sDMA) residues present on the C-terminal tails of SmB, SmD1, and SmD3. This post-translational modification is thought to play a crucial role in snRNP assembly. In human cells, two distinct protein arginine methyltransferases (PRMT5 and PRMT7) are required for snRNP biogenesis. However, in Drosophila, loss of Dart5 (the fruit fly PRMT5 ortholog) has little effect on snRNP assembly, and homozygous mutants are completely viable. To resolve these apparent differences, we examined this topic in detail and found that Drosophila Sm proteins are also methylated by two methyltransferases, Dart5/PRMT5 and Dart7/PRMT7. Unlike dart5, we found that dart7 is an essential gene. However, the lethality associated with loss of Dart7 protein is apparently unrelated to defects in snRNP assembly. To conclusively test the requirement for sDMA modification of Sm proteins in Drosophila snRNP assembly, we constructed a fly strain that exclusively expresses an isoform of SmD1 that cannot be sDMA modified. Interestingly, these flies were viable, and snRNP assays revealed no defects in comparison to wild type. In contrast, dart5 mutants displayed a strong synthetic lethal phenotype in the presence of a hypomorphic Smn mutation. We therefore conclude that dart5 is required for viability when SMN is limiting.

In Chlamydomonas chloroplasts, atpB pre-mRNA matures through a two-step process. Initially, endonuclease cleavage occurs 8 -10 nt downstream of the mature 3 end, which itself lies at the end of a stem-loop-forming inverted repeat (IR) sequence. This intermediate product is then trimmed by a 3 3 5 exonuclease activity. Although the initial endonucleolytic cleavage by definition generates two products, the downstream product of atpB pre-mRNA endonucleolytic processing cannot be detected, even transiently. This product thus appears to be highly unstable, and it can be hypothesized that specific mechanisms exist to prevent its accumulation. In experiments described here, the atpB 3 maturation site was placed upstream of reporter genes in vivo. Constructs containing both the IR and endonuclease cleavage site (ECS) did not accumulate the reporter gene mRNA, whereas constructs containing only the IR did accumulate the reporter mRNA. The ECS alone gave an intermediate result, suggesting that the IR and ECS act synergistically. Additional secondary structures were used to test whether 5 3 3 and/or 3 3 5 exonuclease activities mediated degradation. Because these structures did not prevent degradation, rapid endonucleolytic cleavages most likely trigger RNA destruction after ECS cleavage. On the other hand, fragments resulting from cleavage within the endogenous atpB mRNA could occasionally be detected as antisense transcripts of the adjacent reporter genes. Because endonuclease cleavages are also involved in the 5 maturation of chloroplast mRNAs, where only the downstream cleavage product accumulates, it appears that chloroplast endoribonuclease activities have evolved mechanisms to selectively stabilize different ECS products.Maturation of mRNA can involve multiple steps in both prokaryotic and eukaryotic systems, and results in functional transcripts with a stability and subcellular localization appropriate to their functions. RNA sequence and secondary structure, along with ribonucleases and a variety of accessory factors, are used to achieve these goals. Built into this process is the necessity to recognize nonfunctional RNAs and eliminate them; destruction of nonsense codon-containing mRNAs is a good example of the complexity of such surveillance mechanisms (reviewed in Ref. 1).Our laboratory has focused on 5Ј end and 3Ј end maturation of chloroplast mRNAs, using both vascular plants and the unicellular green alga Chlamydomonas reinhardtii as models. In this respect, chloroplast transcripts have mostly prokaryotic features such as the lack of a trimethylguanosine 5Ј cap, a 3Ј stem-loop-forming inverted repeat (IR) 1 structure, and they are destabilized by polyadenylation (reviewed in Refs. 2 and 3). Processing of chloroplast mRNAs, with rare exceptions, depends on nucleus-encoded proteins, several of which have been identified genetically through screens for plants or Chlamydomonas strains unable to carry out photosynthesis. The phenotypes suggest that defects in intercistronic processing of polycistronic transcripts...

contributed equally to this work Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine± Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (±1 position) and the nucleotide 3¢ to the tRNAf Met anticodon, at position 37. Adenine is conserved at position 37, and a uridine at ±1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA ±1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA fMet . The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added speci®city to transcripts carrying non-standard initiation codons, and/or preserve translational ®delity under certain stress conditions.

The Chlamydomonas reinhardtii chloroplast genome possesses thousands of small dispersed repeats (SDRs), which are of unknown function. Here, we used the petA gene as a model to investigate the role of SDRs in mRNA 3' end formation. In wild-type cells, petA mRNA accumulated as a major 1.3-kb transcript, whose 3' end was mapped to the distal end of a predicted stem-loop structure. To determine whether this stem-loop was required for petA mRNA stability, a series of deletions was constructed. These deletion strains accumulated a variety of petA mRNAs, for which approximate 3' ends were deduced. These 3' ends were found to flank stem-loop structures, many of which were formed partially or completely from inverted copies of SDRs. All strains accumulated wild-type levels of cytochrome f, demonstrating that alternative 3' termini are compatible with efficient translation. The ability to form alternative mRNA termini using SDRs lends additional flexibility to the chloroplast gene expression apparatus and thus could confer an evolutionary advantage.