We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNAmediated RNA silencing in the model algal species, Chlamydomonas reinhardtii. Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon-and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNAtargeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes.

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3′UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas , and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.

27Amyotrophic Lateral Sclerosis (ALS) is an incurable fatal neurodegenerative disease for 28 which the precise mechanisms of toxicity remain unclear despite some significant 29 advances in our understanding of the underlying genetic basis. A holistic, integrated 30 view of cellular changes will be critical to understanding the processes of 31 neurodegeneration and the development of effective treatments. Mutant forms of 32 optineurin (a ubiquitin-binding protein involved in autophagy, membrane trafficking, 33 and NF-kB activation) are found associated with cytoplasmic inclusions containing 34 TDP43 or SOD1 in some ALS patients. We have taken a multi-omics approach to 35 understand the cellular response to OPTN overexpression in a yeast model of ALS. We 36 found that genetic interaction screens and metabolomics provided parallel, highly 37 complementary data on OPTN toxicity. Genetic enhancers of OPTN toxicity in yeast 38 relate directly to the native function of OPTN in vesicular trafficking and intracellular 39 transport, suggesting the human OPTN protein is functional when expressed in yeast 40 even though there is no yeast ortholog. Crucially, we find that the genetic modifiers and 41 the metabolic response are distinct for different ALS-linked genes expressed in yeast. 42This lends strong support to the use of yeast as a model system and omics platform to 43 study ALS. 44 45 46 47 48 Keywords 49 ALS; Systems Biology; Metabolomics 50 51 52 Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease for which 53 there is no effective treatment available. ALS has been divided into familial (FALS) and 54 sporadic (SALS) forms on the basis of family history, with FALS patients accounting 55 for 5% of all ALS cases overall, but this dichotomy is now questioned[1]. Despite our 56 incomplete picture of the genetic landscape of ALS, it is considered a genetic disease. 57 Associated genetic variants, particularly the hexanucleotide repeat expansion of 58 C9orf72[2,3] and mutations in SOD1[4], TDP43[5,6] and FUS[7,8], are the basis for 59 experimental models of ALS in most model systems. Genetic variants and model 60 organism studies implicate a wide range of cellular pathways in the neurodegenerative 61 processes occurring in ALS, including oxidative stress, RNA metabolism, protein 62 aggregation and degradation (autophagy, and the ubiquitin-proteasome system), and 63 intracellular trafficking[9]. The hallmark histopathological feature of ALS is the 64 presence of intracellular protein aggregates. In most cases, these aggregates contain the 65 TDP43 protein, even though mutations in the TDP43 gene are only a rare cause of ALS. 66 A notable exception is patients with SOD1 mutations, where intracellular aggregates 67 contain the SOD1 protein, but not TDP43. With such a complex pathology 68 underpinning ALS, it is vital to develop multi-omics approaches to understand how the 69 interaction of multiple pathways is driving disease progression. 70 71 The OPTN gene encodes Optineurin, a ubiquitin-binding protein ...