Molecular Biology and Genetics MS Thesis Defense by İsa Özdemir

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KOÇ UNIVERSITY

GRADUATE SCHOOL OF SCIENCES & ENGINEERING

MOLECULAR BIOLOGY AND GENETICS

MS THESIS DEFENSE BY İSA ÖZDEMİR

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Title: The roles of miRNAs in proteostasis in C. elegans

Speaker: İsa Özdemir

Time: August 10th, 2017, 14:00

Place: ENG 208

Koç University

Rumeli Feneri Yolu

Sariyer, Istanbul

Thesis Committee Members:

Assist. Prof. Dr. Funda Şar (Advisor, Koc University)

Assoc. Prof. Dr. Nurhan Özlü (Koc University)

Assist. Prof. Dr. Elif Uz (Uludag University)

Abstract:

microRNAs (miRNAs) are  ̴ 22 nt long small non-coding RNAs primarily involved in post-transcriptional gene regulation (PTGR) [1, 2]. The first discovered miRNAs, lin-4 and let-7, are involved in a nematode specific “heterochronic” pathway [3, 4]. Since then, miRNAs are associated with almost all biological processes including apoptosis, cell differentiation and the prevention of numerous disorders [5-7].

Even though the knock-outs of many miRNAs and miRNA families are available, studies on the identification of their roles are still challenging due to the functional redundancies either among the miRNA targets or miRNA family members [8, 9]. In parallel, knock-outs of miRNA biogenesis have been utilized to understand the functions of miRNAs [10]. However, global suppression of miRNA biogenesis is embryonically lethal which hampers the efforts to unravel the post-developmental roles of miRNAs [10]. To overcome this limitation, a conditional temperature‐sensitive (ts) mutant of DGCR8/pash‐1 had been used in C. elegans [11]. Using this allele, post‐developmental expression of miRNAs was shown to be essential for normal life span and physiology in C. elegans [11]. 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 proteostasis. Proteostasis is the process in which the functional proteome has been maintained by an intricate network, called 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. To further question whether the functional decline in PN was the main cause of the defects in pash-1ts adult animals, I enhanced the proteostatic capacity of the animals unable to synthesize miRNAs by Thioflavin T (ThT). The life span of pash-1ts animals were extended by 40% and the motility defects of pash-1ts animals were suppressed by 50-60% upon the 100 mM ThT treatment. In order to specifically address which branches of PN had been affected upon the elimination of miRNAs, I examined the protein folding and degradation systems in pash-1ts animals. Our results indicated that the levels of chaperome was altered upon the elimination of miRNAs suggesting protein folding environment was under the regulation of miRNAs in nematodes. 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. Then 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 also examined the activity of autophagy in the absence of miRNAs. For this, I have knocked-down unc-51 and bec-1 by RNAi and observed that even though lowering the levels of these genes were sufficient to decrease the life span of wild type and pash-1 rescue animals, it did not affect the life span of pash-1ts animals. This suggested that autophagy was already impaired in the absence of miRNAs. To further test this hypothesis, we decided to visualize autophagic vesicles in the animals unable to synthesize miRNAs. Therefore, we have crossed the translational LGG-1 reporter fused with GFP with pash-1ts and pash-1 rescue animals. Our imaging results indicated that even though LGG-1 protein level was higher in pash-1ts animals compared to wild type and pash-1 rescue animals, we observed that less autophagic vesicles were formed in pash-1ts animals relative to wild type and pash-1 rescue animals.

Overall, these results suggested that miRNAs are essential for the regulation of the PN such as protein folding and degradation. In future, miRNAs can be potentially used for diagnostic and therapeutic purposes against the diseases associated with proteostasis defects.