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1、Molecular Cell PreviewsHow Many Ways Are There to Generate Small RNAs?Hailing Jin1,2,* and Jian-Kang Zhu1,2,3,4 1Institute for Integrative Genome Biology2Department of Plant Pathology Halic and Moazed, 2010). miRNAs in plants and animals arise from single-stranded RNA precursors with stem-loop struc
2、- tures, which are processed by the RNase III type endoribonuclease Dicer or Dicer- like (DCL) proteins (Bartel, 2004). While most siRNAs are generated from double- stranded RNAs by Dicers, some siRNAs in C. elegans may be generated directly through de novo synthesis by RNA- dependentRNApolymerases(
3、RDRP) (Ghildiyaland Zamore,2009).The biogen- esis of piRNAs and priRNAs is Dicer- independent and requires other RNA pro- cessing enzymes (Ghildiyal and Zamore, 2009; Halic and Moazed, 2010). Although various classes of miRNAs and siRNAs have been described inanimals and plants, our knowledge of fun
4、- gal small RNAs has been rather limited. No miRNAs were found in fungi. Theonly siRNAs reported in the filamentous fungusNeurosporacrassaareDNA damage induced, the AGO protein QDE- 2-interacting small RNAs (qiRNAs) (Leeet al., 2009). Lee et al. identified several new classes of Neurospora small RNA
5、s that also interact with QDE-2, including miRNA-like RNAs (milRNA) and Dicer- independentsiRNAs(disiRNAs)(Lee et al., 2010). Their analysis revealed several novel small RNA biogenesis path- ways (Figure 1). The study indicates that small RNA biogenesis is much more diverse in fungi than previously
6、thought. By analyzing the small RNAs coim- munoprecipitated with QDE-2 by deep sequencing,Leeetal.identified25 miRNA-like loci that can generate RNA precursors withstem-loopstructures, giving rise to miRNA-like RNAs with a strong strand bias. These milRNAs have a clear preference for U at their 50 t
7、ermini and show a size distribution that peaks at 19 nt and 25 nt. Genetic analysis of four of the most abundant milRNAs (milR-1 to milR-4) suggests that milRNAs havesurprisinglydiversebiogenesispath- ways (Figure 1). The production of milR-1 is Dicer dependent and also requires the activity of a QD
8、E-2-interacting exonu- clease, QIP, and QDE-2 itself. However, QDE-2 slicer activity was not required. QDE-2 binds 33 nt pre-milRNAs in a qip mutant but associated with mature milRNAs in a wild-type strain, suggesting that QDE-2 functions to recruit QIP for milR-1 processing rather than to directlys
9、lice the precursors. The biogenesis of milR-3 is Dicer dependent but does not require QDE-2, so milR-3 biogenesis resembles miRNA formation in plants. AlthoughmilR-4maturationrequiresDicer but not QDE-2 or QIP, milR-4 is reduced but not completely eliminated in the dcl mutant, suggesting a partial d
10、ependence on Dicer and the possible involvement of another endoribonuclease. Most strik- ingly, the biogenesis of milR-2 is Dicer independentandrequirestheslicer activity of QDE-2 but not the QDE-2-inter- actingproteinQIPforitsmaturation.Thus, the fungus N. crassa uses a distinct biogenesis pathway
11、to generate each of these four milRNAs (Figure 1).The finding of Dicer-independent path- ways for milRNA biogenesis prompted Lee et al. to search for new ribonucleases involved in milRNA formation. An RNase III domain-containing protein homologous totheyeastmitochondrialribosomalprotein MRPL3 was id
12、entified as a candi- date for further analysis. A heterokaryotic strain of mrpl3koand wild-type nuclei with reduced mrpl3 mRNA was used because themrpl3deletionstrain islethal. Thelevel ofbothmilR-1andmilR-4butnotofmilR-2 was reduced in the mrpl3ko(het)strain, indi- catingaroleofMRPL3inthebiogenesis
13、of some milRNAs. Although MRPL3s from other eukaryotic organisms are known to be present in thelargesubunit of the mito- chondrial ribosomes, it would be impor- tant to determine the subcellular localiza- tion of Neurospora MRPL3. If the effect of Neurospora MRPL3 on milRNAs biogen- esis is direct,
14、MRPL3 is likely to be func- tional also in the cytoplasm or nucleus.Molecular Cell 38, June 25, 2010 2010 Elsevier Inc.775To test the biological functions of themilRNAs, Lee et al. constructed artifi- cial milRNA targets using a Myc-tagged reporter containing milR-1 complemen- tary sequences, and tr
15、ansformed these constructsintowild-typeandqde-2 mutantstrains.Proteinlevelsofthe reporter were much higher in the qde-2 mutant than in the wild-type, whereas the RNA levels of the reporter were only increased modestly in the qde-2 mutant. These results indicate that like animal miRNAs and some plant
16、 miRNAs, milRNAsinNeurosporamayinduce silencing of their targets mainly by trans- lational inhibition. Using a mammalian miRNA target-prediction program, Leeet al. identified candidate endogenous targets for these milRNAs. Some of the putative target mRNAs of milR-1 were elevated in milR-1koand qde-2 mutants, and some candidate target mRNAs were associated with QDE-2, supporting that these milRNAs do regulate gene expres- sion in vivo. Although the milRNAs aregenerated by different Dicer-depen
