Protein interactions and complexes in human microRNA biogenesis and function

Abstract

Encoded in the genome of most eukaryotes, microRNAs (miRNAs) have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. The aim of this review is to present this process as the integration of a succession of specialized molecular machines exerting well defined functions. The nuclear microprocessor complex initially recognizes and processes its primary miRNA substrate into a miRNA precursor (pre-miRNA). This structure is then exported to the cytoplasm by the Exportin-5 complex where it is presented to the pre-miRNA processing complex. Following pre-miRNA conversion into a miRNA:miRNA* duplex, this complex is assembled into a miRNA-containing ribonucleoprotein (miRNP) complex, after which the miRNA strand is selected. The degree of complementarity of the miRNA for its messenger RNA (mRNA) target guides the recruitment of the miRNP complex. Initially repressing its translation, the miRNP-silenced mRNA is directed to the P-bodies, where the mRNA is either released from its inhibition upon a cellular signal and/or actively degraded. The potency and specificity of miRNA biogenesis and function rely on the distinct protein x protein, protein x RNA and RNA:RNA interactions found in different complexes, each of which fulfill a specific function in a well orchestrated process.

Figure 1

The protein complexes of the microRNA (miRNA)-guided RNA silencing pathway. Both miRNA and messenger RNA (mRNA) encoding genes are transcribed by RNA polymerase II (RNA pol II). Whereas the latter yields a translatable mRNA transcript, the miRNA genes generate a non-translated RNA transcript known as the primary miRNA (pri-miRNA), which is recognized by the microprocessor complex composed of the ribonuclease III (RNase III) Drosha and the accessory DiGeorge syndrome critical region gene 8 (DGCR8) protein. DGCR8 binds to the dsRNA-ssRNA junction at the base of the pri-miRNA and acts as a molecular caliper that mesures the distance of 11 bp, where the two RNase III domains of Drosha form an intramolecular structure with each domain cleaving one strand to produce the miRNA precursor (pre-miRNA). This structure is then exported into the cytoplasm by the Exportin-5 complex in a sequence of events involving RanGTP hydrolysis. The pre-miRNA processing complex, which is composed of Dicer, TRBP and PACT, recognizes and processes the pre-miRNA substrate into a miRNA:miRNA* duplex. The PAZ domain of Dicer has been proposed to act as an anchor for the 3′ hydroxylated 2-nt overhang extremity. The connector helix is responsible of the measurement of the 21–23 bp length from the extremity and determines the positioning at the cleavage site of its two RNase III domains that form, as in Drosha, an intramolecular dimer with a unique catalytic site (36). The resulting miRNA:miRNA* loaded complex then joins the miRNA-containing ribonucleoprotein (miRNP) complex that contains Ago2, after which the miRNA strand is selected. The complementarity of the miRNA to its target brings the miRNP complex on a specific mRNA to negatively regulate it, and ultimately direct it to the P-bodies. These punctuate foci in the cell contain silenced mRNAs, which can be either released from their inhibition upon a cellular signal, or simply degraded.