A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo

Abstract

Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18- to 22-nucleotide tRNA 3' fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA fragment small RNAs.

Figure 1.

Twi12 association with tRNA fragments. (A) The schematics show the strategy of ZZTwi12 transgene knock-in and endogenous TWI12 locus knockout; the latter is evidenced with Southern blot data using NheI-digested genomic DNA at right. The position of the Southern blot probe is indicated in the TWI12 locus schematic with a thin gray line. (B) RNAs copurified with Twi12 in vegetative growth (veg) or after 12 h of starvation (st12) were stained directly with SYBR Gold in comparison with size-selected total RNA from the same cultures. (C) The composition of sRNA library sequencing reads is depicted for reads with either a perfect match to the genome or a single internal mismatch as indicated. (D) Read numbers for tRNA fragments copurified with Twi12 were plotted relative to tRNA gene copy number as an approximation of full-length tRNA abundance. The labels of some data points at left were removed for clarity: mtGluTCC, SecTCA, ThrCGT, mtHisGTG. Pseudo indicates combined reads from the 11 annotated tRNA pseudogenes, and mt indicates mitochondrial.

Figure 2.

Retention of the tRNA 3′ fragment as the tightly bound strand. (A) Blot hybridization of Twi12-bound sRNAs was performed using probes complementary to the 5′ or 3′ end of several abundant tRNAs. Direct SYBR Gold staining of Twi12-bound sRNAs is shown at left for reference. (B) Arrowheads and lines indicate the potential processing positions and detected sRNAs, respectively, for two of the tRNAs from A. The 3′ CCA nucleotides of a mature tRNA are not illustrated. (C) Twi12-associated sRNAs were directly stained after RNP isolation using two different gentle, nondenaturing buffer conditions. (D) Twi12-associated sRNAs were 5′ end-labeled after isolation using stringent purification conditions, with or without prior cross-linking; different concentrations of urea were used for washes prior to RNA extraction. (E–G) Twi12-cross-linked sRNAs washed with 2 M urea were used for blot hybridization to detect specific tRNA fragments (shown in E) and for library construction. The length distribution of sequencing reads (F) is shown for 3′ fragments that match the genome perfectly or have an untemplated 3′ C, CC, or CCA, with the combined nucleotide frequency at each position (G) revealing a 5′ sequence signature.

Figure 3.

Distinct biochemical features of the Twi12-bound and starvation-induced tRNA 3′ fragments. (A–C) Size-enriched total RNA from cells harvested 3 h after initiation of starvation (lanes 1–3) and cross-linked Twi12 sRNAs from vegetative growth (lanes 4–6) were subjected to β elimination or Terminator exonuclease treatment followed by direct staining (A) or blot hybridization (B) to detect a specific tRNA 3′ fragment. The tRNA^Gly 3′ fragment detected by blot hybridization is depicted in C as a solid black line against the dashed full-length tRNA. (D) A speculative model is shown for Twi12 tRNA fragment loading and release. The line drawing is based on previous illustrations of tRNA tertiary structure (Purves et al. 1995).

Figure 4.

Increased tRNA 3′ fragment accumulation upon Twi12 overexpression. (A) ZZTwi12 accumulation in wild-type or transgene-containing cells grown with or without cadmium (Cd²⁺) was determined by immunoblot against the epitope tag at the indicated time points of continuous growth. Cadmium was first administered at time 0 (0.75 μg/mL), and was supplemented again after 12 h (0.3 μg/mL) and 21.5 h (0.5 μg/mL), followed by overnight culture to the maximal cell density of stationary phase. (B,C) Total RNA was harvested and size-enriched from cells in stationary phase (32.5 h), followed by direct staining (B) or blot hybridization (C). Similar results were observed using samples from cells harvested prior to reaching maximum density (data not shown), but stationary-phase culture sRNAs are shown here to allow comparison of Twi12-bound tRNA 3′ fragments and all other tRNA fragment populations, including tRNA halves (see Supplemental Fig. S3).