UC Berkeley

Small RNA (sRNA)-mediated silencing is a strategy used by almost all eukaryotes to regulate gene expression. The only component common to all sRNA-mediated silencing pathways is a PAZ, PIWI domain-containing (PPD) protein, more commonly called Argonaute/Piwi (Ago/Piwi), after the founding members of the family, which have the same domain structure but fall into two subfamilies based on primary sequence similarity. The conserved PAZ and PIWI domains are required for binding the 3' and 5' ends of the sRNA, respectively. The ribonucleoprotein (RNP) complex then sequence-specifically targets nucleic acid substrates through base pairing between the sRNA guide and the target.

Argonaute subfamily proteins and bound sRNAs are ubiquitously expressed in many organisms and have been intensely studied over the past decade. Several mechanisms used by Argonuates to silence gene expression are known in detail. Piwi subfamily proteins and bound RNAs on the other hand are germline-restricted in many organisms and have been intensely studied only for the last five years. They are known to be important for silencing transposons, but detailed mechanisms of their action and other roles are still unknown.

Tetrahymena thermophila expresses eight distinct Piwi-subfamily proteins, no Argonaute subfamily proteins, and no transposons, allowing simplified discovery of other Piwi-bound sRNA classes. I have used protein tagging and affinity purification approaches along with deep sequencing to characterize sRNAs bound to each of these Tetrahymena Piwis (Twis), and have uncovered a great diversity of sRNA classes in this organism (Chapter One). In particular, the only essential Piwi in Tetrahymena, Twi12, specifically binds tRNA fragments (Chapter Two). My further studies of Twi12 have revealed its association with the 5' to 3' exoribonuclease Xrn2 in the nucleus (Chapter Three). In the final chapter I describe biochemical techniques and approaches useful for studies of RNPs in Tetrahymena (Chapter Four).