How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

Abstract

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Feedback regulation of small RNA processing by the cleavage product

“…Under these conditions, accumulation of GlmZ* may limit further processing of full-length GlmZ thereby maintaining a robust GlmS basal level. Interestingly, a similar regulatory scenario has also been proposed for human microRNAs [36]. In this case, miRNAs such as miR-21-5p were shown to inhibit their own biogenesis from the corresponding miRNA precursors, at least in vitro.…”

Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity

“…Previously we have shown that hDicer, apart from the canonical substrates, binds structurally diverse short ssRNA molecules [35][36][37][38]. Consequently, we decided to test whether hDcr and its variants lacking the PAZ domain would cleave short ssRNAs.…”

Ludzka rybonukleaza Dicer – struktura i funkcje biologiczne

“…Wpływają one na zmiany struktury Dicer, uczestniczą w sekwestracji białka lub jego translokacji w obrębie komórki. W ostatnim czasie pojawiają się również doniesienia wskazujące na to, że nie tylko czynniki białkowe, ale również cząsteczki kwasów nukleinowych są zaangażowane w regulację aktywności Dicer [60,72,73]. W komórkach ludzkich oraz C. elegans w obrębie cząsteczek mRNA i lncRNA zidentyfikowano tzw.…”