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MicroRNAs (miRNAs), small-sized ~22 nucleotide noncoding RNA (ncRNA) regulate diverse biological processes, mainly developmental timing and tumorigenesis. Still little is known about miRNAs as potential regulators of membrane trafficking that are crucial for growth and homeostasis.Therefore the aim of this study was to systematically identify miRNAs that regulate protein trafficking and to investigate the mechanism of their action. I carried out high-throughput microscopy-based screen to identify miRNAs playing a role in ER to PM trafficking of secretory cargo tsO45-G. 875 miRNAs were down-regulated by Anti-miR inhibitors. 93 miRNAs facilitated and 41 miRNAs inhibited tsO45-G secretion under these conditions. Down-regulation of endogenously expressed miR-30b facilitated tsO45-G secretion, whereas over- expression of miR-30b inhibited tsO45-G traffic and induced fragmentation of the Golgi complex.Therefore, miR-30b was chosen for further study particularly because it is known to be tumor suppressor. No experimental data of miR-30b targets playing a role in membrane trafficking are available. To identify functionally relevant targets of miR-30b, I used prediction programs,TargetScan, PicTar and miRanda. Out of 444 overlapping targets, 18 core trafficking regulators were selected for RNAi-based ts-O45-G assay. 10 of them recapitulated the inhibition phenotype of over-expressed miR-30b. 3 genes (AP3S1, Golgin-97 and SCAMP1) were validated as targets of miR-30b by luciferase assay, qRT-PCR, Western Blot and mutagenesis. I also showed that combinatorial knockdown of AP3S1, Golgin-97 and SCAMP1 by siRNA inhibits tsO45-G transport to the same degree as that of over-expressed miR-30b. Simultaneous down-regulation of all three genes was needed to rescue the facilitation effect of tsO45-G trafficking induced by Anti-miR-30b. Similar to tsO45-G, transport of yet another basolateral cargo transferrin was inhibited when miR-30b was over-expressed. Interestingly, AP3S1, Golgin-97 and SCAMP1 are known to play a role in between TGN and PM. Indeed, I showed all three genes inhibited tsO45-G transport from trans-Golgi network (TGN) to PM albeit they do not belong to the same protein complex. Our findings suggest that miR-30b regulates TGN or RE to PM trafficking by targeting multiple regulators in concert. In addition, I tested whether one type of long noncoding RNA (lncRNA), namely pseudogenes could act as regulators of membrane trafficking. Here, we reported for the first time that 15 out of 68 RAB GTPases have retroposed transcripts. 3 out of 15 retroposed transcripts (RAB42, RAB5c and RAB28) were shown to be expressed in HeLa cells; and showed varying expression across different cell types. Retroposed transcript of RAB42, named RAB42p inhibited transport of tsO45-G when down-regulated. RAB42p was predicted to have full length ORF showing 92% identical on amino acid level. This branch of my project opens a new research field to investigate whether RAB42p carries out its potential regulatory function of membrane trafficking as a new protein or exerts its action as lncRNA.
|Supervisor:||Nickel, Prof. Dr. Walter|
|Date of thesis defense:||30 January 2014|
|Date Deposited:||12 Feb 2014 07:44|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|
|Controlled Keywords:||miRNAs, secretion, pseudogene|