MikroRNA'ların C. elegans'ta proteostasis üzerine etkisi

microRNAs (miRNAs) are ̴22 nt long small non-coding RNAs involved in post-transcriptional gene regulation (PTGR) [1, 2]. The first discovered miRNAs, lin-4 and let-7, act on a nematode specific "heterochronic" pathway [3, 4]. Since then, miRNAs are associated with a plethora of biological processes including apoptosis, cell differentiation and immune system [5-7]. While studies using the knockouts of miRNA biogenesis genes showed that miRNAs are essential for both embryonic and larval development, it was not possible to address post-developmental roles of miRNAs till a temperature sensitive DGCR8/pash-1 mutant, here after called pash-1ts, was isolated in C. elegans [8]. Using this allele, post‐developmental expression of miRNAs was shown to be essential for normal life span and physiology in C. elegans [8]. However, the underlying mechanisms behind the rapid aging phenotype of pash-1ts animals remained elusive. One of the important hallmarks of aging is the decline in proteostatic capacity. Proteostasis is the process in which the functional proteome has been maintained by proteostasis network (PN), including molecular chaperones, proteasome and autophagy. Defects in PN result in cytotoxic protein aggregations, rapid aging and deterioration of the locomotion in C. elegans. Here, I showed that pash-1ts animals phenocopied the proteostasis defects which could be rescued by the enhancement of proteostasis by Thioflavin T (ThT). Then, I examined how PN was affected in the absence of miRNAs. Our results indicated that the mRNA levels of small heat shock proteins were altered in pash-1ts animals. Of 24 chaperones, sip-1, Q9N350, hsp-25, and hsp-60 were downregulated while F08H9.4a, F44E5.4, F08H9.3 and ZK1128.7 were upregulated in pash-1ts animals compared to wild type and pash-1 rescue animals. Moreover, I have tested the protein degradation systems. First I examined the transcript levels of proteasome genes by quantitative RT-PCR. Interestingly, the mRNA levels of the longevity determining proteasome genes, pbs-5 and rpn-6.1, increased in the absence of miRNAs. However, these have not been phenotypically reflected on pash-1ts animals. Lastly, I have examined the activity of autophagy in the absence of miRNAs. I have knocked-down unc-51 and bec-1 by RNAi and observed that it did not affect the life span of pash-1ts animals, suggesting that autophagy was already impaired in the absence of miRNAs. To validate, we visualized autophagic vesicles using translational LGG-1 reporter fused with GFP. Even though LGG-1 protein level was higher in pash-1ts animals compared to wild type and pash-1 rescue animals, we observed that a decrease in the number of autophagic vesicles formed in pash-1ts mutants. Overall, these results suggested that miRNAs are essential for the regulation of protein folding and degradation. In future, miRNAs can be potentially used for diagnostic and therapeutic purposes against the diseases associated with proteostasis defects.