A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo

New noncoding RNA populations continue to be discovered (Mercer et al. 2009; Siomi and Siomi 2009), implying that there are biological events of RNA-mediated regulation and RNA processing that remain to be appreciated. Beyond the microRNAs, siRNAs, and germline Piwi-interacting RNAs that guide the specificity of Argonaute/Piwi (Ago/Piwi) RNP function (Tolia and Joshua-Tor 2007), the search for novel small RNAs (sRNAs) has uncovered fragments of mature tRNAs that are poorly, nonpreferentially, or not specifically associated with Ago proteins (Cole et al. 2009; Thompson and Parker 2009a; Haussecker et al. 2010). Abundant tRNA fragments resulting from conditionally induced cleavage of the anti-codon loop were first reported in the ciliated protozoan Tetrahymena, and similar tRNA cleavage phenomena have been revealed as a broadly conserved prokaryotic and eukaryotic response to stress or change in developmental state (Lee and Collins 2005; Thompson and Parker 2009a; Garcia-Silva et al. 2010). Stress-induced tRNA cleavage involves a nuclease from the RNase T2 or RNase A family in budding yeast or human cells, respectively, not an RNase III family Dicer enzyme, and therefore generates a 5′ hydroxyl rather than 5′ monophosphate product (Fu et al. 2009; Thompson and Parker 2009b; Yamasaki et al. 2009). Libraries of RNA sequences obtained following Ago/Piwi protein enrichment or size selection of total RNA typically contain a minor fraction of tRNA fragments, which are expected contamination based on high tRNA abundance and the multiple pathways of tRNA degradation (Phizicky and Hopper 2010). Some examples of precursor or mature tRNA processing by Dicer have been reported, one of which occurs by Dicer recognition of an alternative short-hairpin-like fold of the primary transcript (Babiarz et al. 2008; Cole et al. 2009). Recent studies also describe Dicer-independent accumulation of the tRNA 3′ trailer (a primary transcript segment between the tRNA 3′ end and the transcription termination signal), creating sRNAs proposed to regulate cellular proliferation and/or the homeostasis of RNA silencing (Lee et al. 2009; Haussecker et al. 2010). We previously purified tagged versions of eight distinct Tetrahymena Piwi family (Twi) proteins for analysis of sRNAs generated by the Tetrahymena Dicer enzymes Dcl1 and Dcr2. Dcl1 produces ∼28- to 29-nucleotide (nt) sRNAs that mediate heterochromatin formation and DNA elimination in the sexual cycle of reproduction (Malone et al. 2005; Mochizuki and Gorovsky 2005), while Dcr2 produces ∼23- to 24-nt sRNAs involved in gene regulation during asexual growth (Howard-Till and Yao 2006; Lee and Collins 2006, 2007; Couvillion et al. 2009). Of the eight distinct Twi proteins, only Twi12 failed to enrich a profile of sRNAs consistent with the size range for products of Dcl1 or Dcr2 (Couvillion et al. 2009). Surprisingly, among the TWI genes expressed in growing cells, only TWI12 is individually essential (Couvillion et al. 2009; additional data not shown). Because the heterogeneously sized sRNAs bound to Twi12 were previously isolated using Twi12 overexpressed in the presence of competing endogenous protein, they were of uncertain physiological specificity. Here we investigate the specificity of Twi12 sRNA loading.