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. 2006 Jan 1;20(1):28-33.
doi: 10.1101/gad.1377006. Epub 2005 Dec 15.

Two classes of endogenous small RNAs in Tetrahymena thermophila

Suzanne R Lee  1 , Kathleen Collins
Affiliations

Two classes of endogenous small RNAs in Tetrahymena thermophila

Suzanne R Lee et al. Genes Dev. .

Abstract

Endogenous small RNAs function in RNA interference (RNAi) pathways to guide RNA cleavage, translational repression, or methylation of DNA or chromatin. In Tetrahymena thermophila, developmentally regulated DNA elimination is governed by an RNAi mechanism involving approximately 27-30-nucleotide (nt) RNAs. Here we characterize the sequence features of the approximately 27-30-nt RNAs and a approximately 23-24-nt RNA class representing a second RNAi pathway. The approximately 23-24-nt RNAs accumulate strain-specifically manner and map to the genome in clusters that are antisense to predicted genes. These findings reveal the existence of distinct endogenous RNAi pathways in the unicellular T. thermophila, a complexity previously demonstrated only in multicellular organisms.

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Figures

Figure 1.
Figure 1.
Sequence composition and expression profile of the Dicer-related proteins. (A) Schematic of conserved domains in previously characterized Dicers (top) and the T. thermophila Dicers. The less highly conserved RNaseIII domain of DCR1 is denoted in light gray (see Supplementary Figs. S1, S2). The arrow on DCR1 denotes an N-terminal extension relative to DCR2. Bold lines represent regions used for Northern blot probes. (B) Total RNA was used for Northern blot analysis of Dicer expression during the T. thermophila life cycle. Probes for DCR1 and DCR2 mRNAs were used concurrently, followed by probing of the same blot for DCL1 mRNA.
Figure 2.
Figure 2.
Three classes of small RNAs accumulate with distinct life cycle expression profiles. Total RNA was enriched by size filtration for sRNAs from vegetatively growing (3 × 106 cell equivalents), starving (7 h: 3 × 106; overnight: 7.5 × 106), or conjugating (4 h: 1 × 107; 10 h: 7 × 106) cells. The first lane of each triplet set represents column flow-through; the second and third lanes represent first and second washes, respectively. SYBR Gold was used to visualize RNAs.
Figure 3.
Figure 3.
Individual ∼23–24-nt sRNAs accumulate throughout the life cycle with strain-specific expression differences. Total RNA enriched for sRNAs was probed on Northern blots either for an individual sRNA from a single cluster or for sRNAs from all 12 sRNA clusters (sRNA mix) (for actual sRNAs probed, see Supplementary Table 2). The sRNAs 3 and 4 are derived from the same sRNA cluster, while all other sRNAs are derived from distinct clusters. (A) RNA was from SB210 cells in vegetative growth (Veg) or a mix of different time points in starvation (St): 3 h (33%), 6–7 h (58%), and 16–24 h (9%). (B) RNA was from SB210 or CU428 cells in the life cycle stages indicated. Conjugation (Conj) was between SB210 and CU428. (C) RNA was from cells of different strain backgrounds in vegetative growth. Progeny from conjugation were analyzed as a pool before sexual maturity. In B and C, U6 spliceosomal small nuclear RNA served as a loading control.
Figure 4.
Figure 4.
The majority of sRNA clusters are antisense to predicted protein-coding genes. Alignments of three sRNA clusters with annotated genome scaffolds were generated by Gbrowse (see Materials and Methods). The arrows above the scaffold denote the location and orientation of cloned sRNAs, with the number of sRNAs cloned noted in parentheses. (Top) The sRNAs 3 and 4 in Figure 3 map to the cluster on CH445461. (Bottom) The clusters on CH445618 and CH445681 illustrate that within some sRNA clusters, an additional level of sRNA grouping was observed.
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