RNA Journal Club 6/24/10
Stefan L. Ameres, Michael D. Horwich, Jui-Hung Hung, Jia Xu, Megha Ghildiyal, Zhiping Weng, Phillip D. Zamore
Science 328: 1534–1539, 18 June 2010.
This week’s thorough summary and analysis by Anonymous:
Like you, Drosophila siRNAs’d prefer an Argonaute; in fact their Argonaute of choice is Ago2. And this preference comes with a bonus: Hen1 adds 2′-O–methyl groups to all Ago2-bound small RNAs. In this paper, Ameres et al. attempted to uncover the mystery of the methyl group present in the 3’ end of Drosophila siRNAs, which is absent for most microRNAs. Adding an evolutionary twist to the story, in plants, all small RNAs (including microRNAs) are methylated. The obvious difference between the plant microRNAs and those of animals is the degree of complementarity to their targets. In plants, microRNAs are almost completely complementary to their target, hinting that the addition of the methyl group might be related to how tightly the small RNA is bound to its target.
Ameres et al. transfected several Drosophila cell lines with microRNA sensors, which had one or more fully complementary sites for an intended microRNA. Normally these microRNAs are loaded to the Ago1; hence, they lack a methyl group in their 3’ end. They showed that levels of endogenously expressed (miR-34 and bantam) and induced (let-7 and miR-125) microRNAs decreased whence the corresponding microRNA sensor is present. Moreover, for another microRNA (miR-277), which can be loaded into both Ago1 and Ago2, they showed that the Ago1-loaded microRNA population is destabilized upon transfection of a microRNA sensor for miR-277. Therefore, Ago1-loaded small RNAs are prone to destabilization when a fully complimentary target is present.
In order to follow the fate of Ago1-loaded and destabilized microRNAs, the authors radiolabeled the 5’ end of let-7 with 32P in the presence of let-7 sensor in vitro and observed new “tailed” and “trimmed” forms of let-7. They also detected tailed and trimmed forms of endogenously expressed bantam in the presence of a bantam-sensor using Drosophila embryo lysates. An addition of 3’ methyl group protected let-7 from being tailed and trimmed, and absence of Hen1 lead to tailing and trimming of let-7 siRNA. Putting all this evidence into context, the authors conclude that the methyl group protects small RNA from destabilization.
To determine the extent of tolerable complementarity between a microRNA and its target that does not lead to microRNA destabilization, the authors changed the sequence of complementarity site of the microRNA sensor. They found that targets resembling classical microRNA target sites did not result in tailing and trimming, whereas the targets that had less than 8 mismatches to the 3’ end of microRNA triggered destabilization. Furthermore, they also discovered that a small central bulge of 3nt leads to trimming and tailing, but not larger bulges. Hence, target RNA-triggered tailing and trimming require extensive but not necessarily perfect complementarity to the microRNAs.
Since Hen1 plays such a major role in protecting Ago2-bound small RNAs, the authors sequenced the small RNAs from hen1 mutant flies. The abundance and the length of microRNAs were not affected by lack of functional Hen1, but endo-siRNAs showed drastic changes in their lengths and abundance. The authors analyzed the content of the tails that arose due to absence of Hen1 and they found that the tail was either a single adenine or a single or multiple uridines, which is a mark of RNA turnover. Lastly, the authors also looked for target RNA-directed trimming and tailing in mammalian cells and confirmed the presence of a similar mechanism with slight modifications.
The Zamore Lab previously characterized RNA sorting in Drosophila, showing that the degree of complementarity between the small RNA and its star strand was responsible for being selectively loaded into Ago1 or Ago2. MicroRNAs are generally loaded into Ago1 to repress target translation and decrease target stability, often mediated by seed-paring between the microRNA and its target. The presence of highly complementary targets for Ago1 loaded small RNAs lead to remodeling of the small RNA by tailing or trimming. Therefore, Ago1 bound small RNAs are specialized in regulating partially complementary sites, which explain why microRNA targets lack extensive complementarity. The authors also speculate that extensive 3’ pairing might result in release of the small RNA from the PAZ domain of Argonaute, which in turn exposes the small RNA to the nucleotidyl transferases and 3’-to-5’ exonuclease enzymes.
Unfortunately we do not know which factors lead to target RNA-directed tailing and trimming and this will be an interesting venue of research in upcoming years. Moreover, the current model fails to explain the existence of highly complementary microRNA target sites present in both mammals and flies. But the paper formulates the question it tries to answer clearly from the beginning and delivers a satisfactory answer.
Citation for researchblogging.org:
Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z, & Zamore PD (2010). Target RNA-directed trimming and tailing of small silencing RNAs. Science, 328 (5985), 1534-9 PMID: 20558712