Loading and pre-loading processes generate a distinct siRNA population in Tetrahymena

Abstract

The various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact their sorting into different Argonaute proteins in diverse eukaryotes. The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5' end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5' uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3' terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo.

Keywords: Argonaute; Dicer; Tetrahymena; siRNA.

Fig. 1

Analysis of Twi1p-associated small RNAs. (A) Size distribution of the Twi1p-associated RNAs from the wild-type cells. The number of sequenced RNAs (reads per million sequences (RPM)) of each size is shown. (B) Base composition of 29-nt scnRNAs. (C) Base composition of the 29-nt RNAs derived from rRNAs or tRNAs. (D) Deduced structure of the 29-nt scnRNAs before loading. (E) Fraction of the RNAs with uracil as the first base (5′ U). (F) Fraction of the RNAs with adenine as the third base from the 3′ end (-3A). (G) Thermodynamic stability differences between the two ends of the modeled scnRNA duplexes. The thermodynamic stability of the 4 base pairs containing the 5′ end of the guide (ΔGg) and the passenger (ΔGp) strand (see (D)) of the individual modeled scnRNA pairs were calculated, and the differences (ΔΔG = ΔGg − ΔGp) are shown as a histogram.

Fig. 2

Analyses of unloaded scnRNAs and the nucleotide specificity loop. (A) Size distribution of the small RNAs from the TWI1 KO cells. The number of sequenced RNAs (reads per million sequences (RPM)) of each size is shown. (B) Base composition of the 29-nt RNAs. (C) Fraction of the RNAs with uracil as the first base (5′ U). (D) Fraction of the RNAs with adenine as the third base from the 3′ end (-3A). (E) Comparison of the nucleotide specificity loops of the different Argonaute proteins. The region corresponding to the proposed nucleotide specificity loop of human AGO2 is marked with a double-headed arrow. Human (Hs) AGO2 and C. elegans (Ce) Alg-1, which tend to bind to 5′ U/A RNAs, share a conserved sequence in the loop. Tt: Tetrahymena thermophila; Dm: Drosophila melanogaster; At: Arabidopsis thaliana. (F) Fraction of the 5′ U RNAs in HA-Twi1p-associated (black), HA-Twi1p-hAGO2Lmut-associated (gray), or total TWI1 KO (white, same data as (C)) small RNAs. The replaced nucleotide specificity loop region is shown in red. The extra glycine inserted into the nucleotide specificity loop of human AGO2 is marked with a square. (G) Base composition of the non-5′ U 29-nt RNAs from the TWI1 KO cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Analysis of the 3′ U bias of scnRNAs. (A) Fraction of the RNAs from the TWI1 KO cells with uracil as the last base (3′ U). (B) Analysis of the potential trimming and tailing reactions on the 29-nt RNAs from the TWI1 KO cells. The 29-nt RNAs from the TWI1 KO cells were mapped to the 100-kb LMR genomic locus by allowing for two base mismatches and were analyzed to determine the positions at which the mismatches were located. (C) Fraction of the 24- to 32-nt RNAs from the TWI1 KO cells with mismatches in the LMR locus. The 24- to 32-nt RNAs from the TWI1 KO cells were analyzed as in (B), and the average frequency of mismatches at the first base to the second base from the end (purple) and at the last base (gray) are shown. (D) Fraction of the RNAs from the TWI1 KO cells with adenine as the second base from the end (-2A) is shown in green. Expected fraction of -2A RNAs, if scnRNA processing occurs sequentially (this is equal to the fraction of 3′U RNAs in (A)), is shown in gray. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)