MicroRNAs (miRNAs) are ∼21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

SPL3, SPL4 and SPL5 (SPL3/4/5) are closely related members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE family of transcription factors in Arabidopsis, and have a target site for the microRNA miR156 in their 3 ′ UTR. The phenotype of Arabidopsis plants constitutively expressing miR156-sensitive and miR156-insensitive forms of SPL3/4/5 revealed that all three genes promote vegetative phase change and flowering, and are strongly repressed by miR156. Constitutive expression of miR156a prolonged the expression of juvenile vegetative traits and delayed flowering. This phenotype was largely corrected by constitutive expression of a miR156-insensitive form of SPL3. The juvenile-to-adult transition is accompanied by a decrease in the level of miR156 and an increase in the abundance of SPL3 mRNA. The complementary effect of hasty on the miR156 and SPL3 transcripts, as well as the miR156-dependent temporal expression pattern of a 35S::GUS-SPL3 transgene, suggest that the decrease in miR156 is responsible for the increase in SPL3 expression during this transition. SPL3 mRNA is elevated by mutations in ZIPPY/AGO7, RNA DEPENDENT RNA POLYMERASE 6 (RDR6) and SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that it is directly or indirectly regulated by RNAi. However, our results indicate that RNAi does not contribute to the temporal expression pattern of this gene. We conclude that vegetative phase change in Arabidopsis is regulated by an increase in the expression of SPL3 and probably also SPL4 and SPL5, and that this increase is a consequence of a decrease in the level of miR156.

Higher plants undergo a transition from a juvenile to an adult phase of vegetative development prior to flowering. Screens for mutants that undergo this transition precociously produced alleles of two genes required for posttranscriptional gene silencing (PTGS)—SUPPRESSOR OF GENE SILENCING3 (SGS3) and SUPPRESSOR OF GENE SILENCING2(SGS2)/SILENCING DEFECTIVE1 (SDE1)/RNA-DEPENDENT POLYMERASE6 (RDR6). Loss-of-function mutations in these genes have a phenotype similar to that of mutations in the Argonaute gene ZIPPY (ZIP). Epistasis analysis suggests that ZIP, SGS3, SGS2/SDE1/RDR6, and the putative miRNA export receptor, HASTY (HST), operate in the same pathway(s). Microarray analysis revealed a small number of genes whose mRNA is increased in ZIP, SGS3, and SGS2/SDE1/RDR6 mutants, as well as genes that are up-regulated in SGS3 and SGS2/SDE1/RDR6 mutants, but not in ZIP mutants. One of these latter genes (At5g18040) is silenced posttranscriptionally in trans by the sRNA255 family of endogenous, noncoding, small interfering RNAs (siRNAs). The increase in At5g18040 mRNA in SGS3 and SGS2/SDE1/RDR6 mutants is attributable to the absence of sRNA255-like siRNAs in these mutants. These results demonstrate a role for endogenous siRNAs in the regulation of gene expression, and suggest that PTGS plays a central role in the temporal control of shoot development in plants.

The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3′ fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5′ cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway.

In mammalian cells, the nuclear export receptor, Exportin 5 (Exp5), exports pre-microRNAs (pre-miRNAs) as well as tRNAs into the cytoplasm. In this study, we examined the function of HASTY (HST), the Arabidopsis ortholog of Exp5, in the biogenesis of miRNAs and tRNAs. In contrast to mammals, we found that miRNAs exist as single-stranded 20- to 21-nt molecules in the nucleus in Arabidopsis . This observation is consistent with previous studies indicating that proteins involved in miRNA biogenesis are located in the nucleus in Arabidopsis . Although miRNAs exist in the nucleus, a majority accumulate in the cytoplasm. Interestingly, loss-of-function mutations in HST reduced the accumulation of most miRNAs but had no effect on the accumulation of tRNAs and endogenous small interfering RNAs, or on transgene silencing. In contrast, a mutation in PAUSED (PSD), the Arabidopsis ortholog of the tRNA export receptor, Exportin-t, interfered with the processing of tRNA-Tyr but did not affect the accumulation or nuclear export of miRNAs. These results demonstrate that HST and PSD do not share RNA cargos in nuclear export and strongly suggest that there are multiple nuclear export pathways for these small RNAs in Arabidopsis .