Understanding RNA transport via exosomes and SIDT2
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2023-02-20
© 2018 Dr Marilou Barrios
The discovery that RNA not only functions as an intermediate between a gene and a protein but also possesses regulatory functions has brought significant interest in its potential role in development and disease, and it is now apparent that regulatory RNAs are important for the regulation of multiple biological processes. What is less clear, however, is the role of RNAs as intercellular signaling molecules and the mechanisms by which RNAs are transported between cells. One mechanism that has received significant attention involves exosomes, which are nanoscale vesicles released by multiple cell types. While exosomes have been shown to contain regulatory RNAs and to mediate intercellular communication, how RNAs are transferred into exosomes remains poorly understood. One protein that has been recently implicated in RNA transfer is the endo-lysosomal protein SIDT2. While it appears to be important in the transport of viral RNAs for innate immune recognition, SIDT2’s role in trafficking endogenous RNAs remains to be investigated. In this thesis, after providing a general introduction and outlining my relevant methods in the first two chapters, I describe in Chapter 3 how I profiled exosomal RNAs from mouse dendritic cells (DCs) using RNA-Seq and identified a long noncoding RNA, VL30, which is highly enriched in exosomes. Having observed that VL30 lncRNA can be transferred to recipient cells both in vitro and in vivo, my bioinformatic analysis revealed that exosome-enriched isoforms of VL30 lncRNA contain a repetitive motif. Subsequent experiments showed that the motif itself is efficiently incorporated into exosomes, suggesting the possibility that it might directly promote exosomal loading and be useful in future efforts to selectively load therapeutic RNAs into exosomes for clinical use. However, the repetitive motif is predicted to fold into a long double-stranded RNA (dsRNA) hairpin and, consistent with this, its overexpression was associated with induction of a type I interferon response and cell death. In Chapter 4, I explored the role of the dsRNA transporter SIDT2 in incorporating RNAs into exosomes. By confocal microscopy, I showed that SIDT2 co-localises with the exosomal marker CD63, suggesting the possibility that SIDT2 might promote exosomal RNA loading. However, my subsequent studies failed to demonstrate a role for SIDT2 in influencing either the microRNA or long RNA content of exosomes. In Chapter 5, motivated by a desire to understand the possible endogenous RNA substrates of SIDT2, I explored the role of SIDT2 in skeletal muscle. I observed that mice deficient in SIDT2 develop a skeletal muscle myopathy and die prematurely, and that loss of SIDT2 leads to impairment of autophagy, with an accumulation of autophagic vacuoles. If and how the RNA transport function of SIDT2 relates to the impairment of autophagy remains unclear, but interestingly I found that in the absence of SIDT2 endogenous dsRNA accumulates in muscle fibres, leading to the induction of a type I interferon response and apoptosis, with selective loss of fast 2B myofibers and a corresponding reduction in muscle force and grip strength. Finally, in chapter 6, I investigated the effects of SIDT2 in an RNA editing-deficient mouse model that carries the ADAR1 E861A loss-of-function allele. These mice accumulate endogenous dsRNA due to their inability to perform RNA editing of dsRNA substrates, and die during embryonic life due to a massive type I interferon response. Given that loss of SIDT2 has been found to modulate the interferon response by trafficking viral dsRNA, I hypothesised that loss of SIDT2 might modulate the phenotype of the ADAR1E861A/E861A mice, but found this not to be the case.
KeywordsRNA transport; exosomes; Sidt2; ADAR1
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