MicroRNAs (miRNAs) are small regulatory RNAs, ~22 nucleotides (nt) in length that are typically derived from a single arm of imperfect, ~80-nt long RNA hairpins located within polymerase II (pol II)-derived transcripts referred to as primary miRNAs (pri-miRNAs) (Fig. 1). Pri-miRNAs are capped and polyadenylated and may be almost any size, ranging from hundreds to thousands of nucleotides, and may encode a single miRNA or a cluster of several miRNAs. Pri-miRNA stem-loops, which consist of an ~33-base pair (bp) imperfect stem and a ≥10-nt terminal loop, are cleaved by the RNase III enzyme Drosha, acting together with its co-factor DGCR8, ~22 bp from the stem/loop junction, thereby excising the ~60-nt precursor miRNA (pre-miRNA) hairpin. Since the flanking 5’ and 3’ arms of the pri-miRNA are degraded following Drosha cleavage, pre-miRNAs are typically found within the exonic regions of non-coding RNAs or in the introns of protein coding or non-coding transcripts (Fig. 1).
Cleavage by Drosha leaves the pre-miRNA hairpin with a 2-nt 3’ overhang. This is recognized by Exportin 5, which transports the pre-miRNA to the cytoplasm. There, the same 2-nt 3’ overhang is recognized by Dicer, another RNase III enzyme, and its co-factor TRBP. Binding of Dicer/TRBP to the base of the pre-miRNA is followed by cleavage to release the terminal loop, yielding an RNA duplex of ~20 bp flanked by 2-nt 3’ overhangs (Fig. 1). The RNA strand that is less tightly base paired at the 5’ end is loaded into the RNA induced silencing complex (RISC) and forms the mature miRNA. The miRNA then guides RISC to mRNAs bearing complementary target sites.
Although the vast majority of miRNAs are generated as described above, some exceptions exist. For example, some miRNAs are derived from short, excised introns, called miRtrons, which resemble pre-miRNA hairpins and only require Dicer for maturation. The miRNAs encoded by the murine γ-herpesvirus 68 (MHV68) are currently unique in that they are transcribed by RNA polymerase III to generate tRNA:pre-miRNA fusion transcripts that are then cleaved by tRNAse Z, which liberates the pre-miRNA for nuclear export by Exportin 5. These viral miRNAs are therefore similar to miRtrons in that they are also produced in the absence of Drosha but require processing by Dicer.
RISCs, which are minimally composed of a mature miRNA an Argonaute (Ago) protein and a member of the GW182 protein family, usually but not invariably bind to the 3’ untranslated region (3’UTR) of targeted transcripts. Functional targets are generally fully complementary to nucleotides 2-7, preferably 2-8, at the 5’ end of the miRNA, referred to as the miRNA seed. There are a few examples known, however, where base pairing between the target transcript and the 3’ half of the miRNA can compensate for mismatches in the seed. Perhaps the best example of this phenomenon is provided by the miRNA lin-4 and a well-characterized target mRNA encoding lin-14, where robust inhibition of lin-14 expression is conferred by target sites lacking full seed homology.
RISCs bound to partially complementary mRNA targets induce the translational repression of that mRNA, by a mechanism that remains to be fully defined (Fig. 1).Translational repression in turn often induces a modest destabilization of the target mRNA. RISCs tend to function in a cooperative manner; the greater the number of RISCs bound to a target transcript, the greater the inhibitory effect.
While RISCs bound to partially complementary mRNA targets primarily induce translational inhibition, mRNAs bearing perfectly complementary miRNA targets are cleaved by RISC and degraded in a process often referred to as RNA interference (RNAi) (Fig. 1). Unlike translational inhibition, which requires stable binding of RISC to its target mRNA, RNAi permits a single RISC to act enzymatically to irreversibly inhibit the expression of multiple mRNA molecules.
miRNAs are expressed by plants and metazoans where they have been found to play a role in a wide variety of cellular processes including cellular proliferation, regulation of development, apoptosis, homeostasis and tumor formation. Humans encode over 700 different miRNAs, which tend to be expressed in a developmental or tissue-specific manner. It has been demonstrated that individual miRNAs are capable of directly down-regulating the expression of hundreds of different mRNAs, and over 30% of all mammalian mRNAs are thought to be regulated by miRNAs.
In addition to the cells of multicellular organisms, we now know that many pathogenic viruses, in particular viruses belonging to the herpesvirus family, also express miRNAs in infected cells. These miRNAs are known to antagonize innate immune responses, regulate viral latency and modulate the growth and survival of infected cells. The Cullen laboratory focuses on understanding how viral miRNAs regulate viral replication and pathogenesis, using HSV-1 and EBV as model systems. We also are investigating how cellular miRNAs affect virus replication, using HIV-1 and EBV as model systems.