MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficiency. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development.

MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that was recently implicated in miRNA processing. Here we show that human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript.

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the posttranscriptional level 1 . Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex2 -4. These pre-miRNAs are cleaved by the RNase III Dicer5 -8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence9. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein10), which contains three double-stranded, RNAbinding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2)11 , 12, the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.To gain an insight into the components of the miRNA/siRNA processing machinery, we isolated a Dicer-containing complex from human cells. This was accomplished by developing HEK293-derived stable cell lines expressing Dicer tagged with Flag (Flag-Dicer). Flag-Dicer was isolated using affinity chromatography, and the affinity eluate was subjected to SDSpolyacrylamide gel electrophoresis (PAGE) followed by silver staining and western blot analysis.Western blot and mass spectrometric analyses indicated that most polypeptides in the Dicer affinity eluate were products of the proteolytic break down of Dicer (Fig. 1a). However, mass spectroscopy identified a 50-kDa band (six peptide sequences that migrated slightly above the contaminating MEP50 band) corresponding to the human immunodeficiency virus (HIV)-1 transactivating response (TAR) RNA-binding protein (TRBP) 10 . The TRBP gene encodes a protein with three double-stranded RNA-binding domains (dsRBDs). Analysis of the nonredundant protein database by Blast identified proteins with close homology to TRBP in both vertebrates and Drosophila (CG6866) (Fig. 1b) (Fig. 1c). The presence of Dicer was also confirmed by mass spectrometric sequencing. Moreover, additional bands (indicated with an asterisk in Fig. 1c) correspond to SKB1 and MEP50, common contaminants of Flag purification. Although most of TRBP eluted in smaller fractions (32 and beyond; perhaps as a consequence of overexpression), a minor portion of TRBP eluted as a large complex (fractions 16 and 18) not easily visualized by silver sta...

METTL3 is a RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through N6-methyladenosine (m6A) modification. Here we find that METTL3 promotes translation of certain mRNAs including epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ in human cancer cells. In contrast to current models that invoke m6A reader proteins downstream of nuclear METTL3, we find METTL3 associates with ribosomes and promotes translation in the cytoplasm. METTL3 depletion inhibits translation, and both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA. Mechanistically, METTL3 enhances mRNA translation through an interaction with the translation initiation machinery. METTL3 expression is elevated in lung adenocarcinoma and using both loss- and gain-of-function studies we find that METTL3 promotes growth, survival, and invasion of human lung cancer cells. Our results uncover an important role of METTL3 in promoting translation of oncogenes in human lung cancer.