As more genome sequences become available with every passing day, it is apparent that not all of it codes for useful proteins. Much of eukaryotic genomes are composed of transposons, parasitic elements that propagate in a cell. Their success impacts host genome integrity and shapes gene expression. However, genetic analysis reveals that most transposons are quiescent and non-duplicating. This is achieved by silencing pathways that manage to keep transposons in an inactive state under normal conditions. Chief among them is RNA interference, a small RNA guided mechanism that suppresses the expression of complementary transcripts. RNAi acts as a nucleic acid based immune system against viruses and repetitive sequences that might otherwise cause genome instability. The diversity of RNAi directed silencing pathways is matched only by the diversity of transposons. In many organisms, RNAi cooperates with heterochromatin factors to target repeats clustered in specific genomic regions like centromeres for small RNA generation leading to RNAi-mediated transcriptional silencing. In this work, we report two mechanisms of post-transcriptional silencing of transposable elements that are independent of the heterochromatin machinery as well as the preliminary characterization of two new RNAi-related components using the model fungus, Cryptococcus neoformans.
Chapter 2 of this thesis focuses on the ‘multi-point lock’ involving mRNA degradation and translation silencing effected by RNAi on an active DNA element in C. neoformans. This sheds light on a transposon suppression pathway that is capable of severely limiting transposon mobilization in a manner independent of genomic location.
Chapter 3 discusses an attempt to discover additional components of the RNAi-mediated transposon silencing pathway through the application of a trans-kingdom insertional mutagenesis screen in a transposon activity reporter. We outline the identification of Sqs1, a spliceosomal discard related factor as a strong positive hit in the screen such that small RNA accumulation is completely abolished in the mutant. Further genetic and proteomic studies hint at the involvement of the spliceosome release machinery in the coupling of the two important biological processes during small RNA biogenesis.
In Chapter 4, we characterize an Argonaute-associated factor that might play a role in the bi-fold silencing pathway described in Chapter 2. Argonaute proteins are essential components of the RNA induced silencing complex (RISC). We isolated Ago1 binding proteins in C. neoformans and identified a protein called Gwo1 that contains numerous GW/WG dipeptide repeats common to Ago-associated proteins in higher organisms. It bears other hallmarks of GW-proteins playing a role in the miRNA pathway such as P-body localization but is absolutely required for transposon silencing. Thus, Gwo1 might act as the missing link connecting Ago1 to mRNA degradation/ translational silencing of transposable elements.
These studies uncover novel pathways and proteins that are critical for RNAi-mediated silencing of transposable elements. This might offer insight into similar strategies that are operating in other eukaryotes.
