In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) play important roles as intermediates. Primary siRNAs are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer; in some organisms, secondary siRNAs are also produced by processes involving RNA-dependent RNA polymerases (RdRPs), which act on target mRNAs. Using a cell-free assay system prepared from Caenorhabditis elegans, we analyzed the production and activity of secondary siRNAs. In this cell-free system, RdRP activity acts on mRNA-derived templates to produce small RNAs. The RRF-1 complex is predominantly responsible for the RdRP activity, and synthesizes secondary-type siRNA molecules in a Dicerindependent manner. Notably, secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Secondary rather than primary siRNAs may play a major role in the destabilization of target transcripts during RNAi in C. elegans.

The positive regulatory machinery in the microRNA (miRNA) processing pathway is relatively well characterized, but negative regulation of the pathway is largely unknown. Here we show that a complex of nuclear factor 90 (NF90) and NF45 proteins functions as a negative regulator in miRNA biogenesis. Primary miRNA (pri-miRNA) processing into precursor miRNA (pre-miRNA) was inhibited by overexpression of the NF90 and NF45 proteins, and considerable amounts of pri-miRNAs accumulated in cells coexpressing NF90 and NF45. Treatment of cells overexpressing NF90 and NF45 with an RNA polymerase II inhibitor, ␣-amanitin, did not reduce the amounts of pri-miRNAs, suggesting that the accumulation of pri-miRNAs is not due to transcriptional activation. In addition, the NF90 and NF45 complex was not found to interact with the Microprocessor complex, which is a processing factor of primiRNAs, but was found to bind endogenous pri-miRNAs. NF90-NF45 exhibited higher binding activity for pri-let-7a than pri-miR-21. Of note, depletion of NF90 caused a reduction of pri-let-7a and an increase of mature let-7a miRNA, which has a potent antiproliferative activity, and caused growth suppression of transformed cells. These findings suggest that the association of the NF90-NF45 complex with pri-miRNAs impairs access of the Microprocessor complex to the pri-miRNAs, resulting in a reduction of mature miRNA production.MicroRNAs (miRNAs) constitute a class of noncoding small RNAs that function as repressors for eukaryotic gene regulation by binding to the 3Ј untranslated regions of target mRNAs (2). This binding causes mRNA cleavage or translational inhibition of the mRNA, depending upon the degree of complementarity. The lengths of miRNAs are 21 to 23 nucleotides (nt), and over 500 miRNAs have been discovered in mammals. miRNAs regulate the expression of a large number of genes (38) that are involved in cell proliferation, apoptosis, hematopoietic differentiation, viral infection, and tumorigenesis (4,5,7,22,26,32,39,45).In mammals, miRNA genes are transcribed by RNA polymerase II as primary miRNAs (pri-miRNAs) (36). These primiRNAs are processed into precursor miRNAs (pre-miRNAs) by the Microprocessor complex (8,13,20,31,33). Another complex comprised of exportin-5 and RanGTP transports the pre-miRNAs from the nucleus to the cytoplasm (3, 40, 58). In the cytoplasm, Dicer, a cytoplasmic RNase III enzyme, cleaves the pre-miRNAs to approximate 22-nt mature miRNA duplexes with 2-nt 3Ј overhangs (14,24,28). One strand of the duplex is incorporated into the RNA-induced silencing complex (12,19,29,41,51). The single strand of RNA guides the RNA-induced silencing complex to the target mRNA with sequence complementarity, which leads either to mRNA cleavage or to translational repression (12,24,41,44).The Microprocessor complex, which cleaves pri-miRNA to pre-miRNA during miRNA biogenesis, is comprised of a nuclear RNase III enzyme, Drosha, and its cofactor, DGCR8 (8,13,20). In addition to the Microprocessor complex, excessively expressed Drosha forms ...

Edited by Michael IbbaKeywords: RNA-directed immunoprecipitation Proteomics LAPP1 Poly(A) tail 5 0 TOP mRNA a b s t r a c t A poly(A) tail functions in mRNA turnover and in facilitating translation as a ribonucleoprotein complex with poly(A) binding proteins (PABPs). However, factors that associate with the poly(A) tail other than PABPs have not been described. Using proteomics, we identified candidate proteins that interact to the 3 0 terminus of the poly(A) tail. Among these proteins, we focused on La motif-related protein 1 (LARP1) and found that LARP1 specifically recognizes the 3 0 termini of normal poly(A) tails. We also reveal that LARP1 stabilizes multiple mRNAs carrying 5 0 terminal oligopyrimidine tract (5 0 TOP). Our findings suggest that LARP1 may be involved in the post-transcriptional regulation of gene expression, at least in several 5 0 TOP mRNAs, through the binding to 3 0 terminus of the poly(A) tail.