Translation inhibition is a major but poorly understood mode of action of micro(mi)RNAs in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here we show that the translation inhibition but not the mRNA cleavage activity of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild type and the amp1 mutant for membrane-bound but not total polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.

Intergenic transcription by RNA Polymerase II (Pol II) is widespread in plant and animal genomes, but the functions of intergenic transcription or the resulting noncoding transcripts are poorly understood. Here, we show that Arabidopsis Pol II is indispensable for endogenous siRNA-mediated transcriptional gene silencing (TGS) at intergenic low-copy-number loci, despite the presence of two other polymerases-Pol IV and Pol V-that specialize in TGS through siRNAs. We show that Pol II produces noncoding scaffold transcripts that originate outside of heterochromatic, siRNA-generating loci. Through these transcripts and physical interactions with the siRNA effector protein ARGONAUTE4 (AGO4), Pol II recruits AGO4/siRNAs to homologous loci to result in TGS. Meanwhile, Pol II transcription also recruits Pol IV and Pol V to different locations at heterochromatic loci to promote siRNA biogenesis and siRNA-mediated TGS, respectively. This study establishes that intergenic transcription by Pol II is required for siRNA-mediated TGS, and reveals an intricate collaboration and division of labor among the three polymerases in gene silencing.[Keywords: RNA polymerase; siRNA; DNA methylation; transcriptional gene silencing; noncoding transcripts] Supplemental material is available at http://www.genesdev.org.

Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5′ monophosphate, lack a poly(A) tail at the 3′ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants.

Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs – production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER.DOI: http://dx.doi.org/10.7554/eLife.22750.001

SUMMARYThe architecture of the rice inflorescence, which is determined mainly by the number and length of primary and secondary inflorescence branches, is of importance in both agronomy and developmental biology. The position and number of primary branches are established during the phase transition from vegetative to reproductive growth, and several of the genes identified as participating in this process do so by regulating the meristemic activities of inflorescence. However, little is known about the molecular mechanism that controls inflorescence branch elongation. Here, we report on a novel rice mutant, short panicle1 (sp1), which is defective in rice panicle elongation, and thus leads to the short-panicle phenotype. Gene cloning and characterization indicate that SP1 encodes a putative transporter that belongs to the peptide transporter (PTR) family. This conclusion is based on the findings that SP1 contains a conserved PTR2 domain consisting of 12 transmembrane domains, and that the SP1-GFP fusion protein is localized in the plasma membrane. The SP1 gene is highly expressed in the phloem of the branches of young panicles, which is consistent with the predicted function of SP1 and the sp1 phenotype. Phylogenetic analysis implies that SP1 might be a nitrate transporter. However, neither nitrate transporter activity nor any other compounds transported by known PTR proteins could be detected in either a Xenopus oocyte or yeast system, in our study, suggesting that SP1 may need other component(s) to be able to function as a transporter, or that it transports unknown substrates in the monocotyledonous rice plant.