Cleaving with preferences

In eukaryotes, RNA interference-derived small RNAs (sRNAs) play a critical role in development, gene expression, and genome stability. Dicer proteins have an important role as well, as they produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. The enzymes are best known for their role as endoribonucleases in small RNA pathways, where they regulate biogenesis of a wide range of small RNAs derived from various sources.

Up to now though, Dicer proteins have never been shown to have sequence cleavage preferences. In a new Cell paper, Cristina Hoehener, Iris Hug, and Mariusz Nowacki from the Institute of Cell Biology of the University of Bern now report three Dicer-like enzymes in Paramecium with pronounced sequence cleavage preferences. These preferences lead to the production of RNAs precisely matching ends of transposon-derived short DNA elements with corresponding end base preferences. The researchers propose a biological role for these newly characterised enzymes and their sequence-biased cleavage products. Through in vitro assays they tested whether the sequence specificity is a consequence of a specific Dicer cleavage, or as in other organisms, due to selection by Argonaute proteins. They could exclude the lat- ter and were able to show that Paramecium Dicer-like proteins have sequence specificity for their target dsRNA, which contributes to the efficiency and precision of the DNA elimination machinery.

The observed features make Paramecium Dicers more similar to restriction enzymes, although their sequence requirements are not as stringent as in the case of bacterial restriction endonucleases cleaving dsDNA. It remains to be determined how the Paramecium Dicer-like enzymes recognize the dsRNA target in a sequence-specific manner.

As for the biological role, the researchers propose the facilitation of the precise elimination of germline-specific DNA. The Nowacki group had previously reported an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs in targeting DNA for elimination. They suggest that the Dicer-like proteins evolved together with the excision machinery to be able to efficiently eliminate those DNA elements from the ge- nome. Analysis of developmental-specific sRNAs in Paramecium had already shown that the sRNAs map preferentially to the very ends of IESs. The researchers therefore believe that sequence specific Dicer proteins have evolved to produce sRNAs that help recognizing the ends of excised DNA. This mechanism will ensure that IES ends are marked precisely by small RNAs and target their elimination in a precise fashion. This allows to optimize the mechanism of RNA-guided DNA elimination by only producing sRNAs that are useful. More importantly, it ensures production of small RNAs marking the DNA ends very precisely, which may be essential, because most IESs are located within coding regions of the genome.

As a side note the researchers suggest that the ability of Dicer-like proteins to cleave at specific sequences within a long dsRNA template could provide an opportunity for future protein engineering to produce enzymes with unique sequence preferences.

Hoehener C. et al. (2018) Cell, 173(1), 234- 247 (open access)

By Roland Fischer