The ASYMMETRIC LEAVES Complex Employs Multiple Modes of Regulation to Affect Adaxial-Abaxial Patterning and Leaf Complexity.

Flattened leaf architecture is not a default state but depends on positional information to precisely coordinate patterns of cell division in the growing primordium. This information is provided, in part, by the boundary between the adaxial (top) and abaxial (bottom) domains of the leaf, which are specified via an intricate gene regulatory network whose precise circuitry remains poorly defined. Here, we examined the contribution of the ASYMMETRIC LEAVES (AS) pathway to adaxial-abaxial patterning in Arabidopsis thaliana and demonstrate that AS1-AS2 affects this process via multiple, distinct regulatory mechanisms. AS1-AS2 uses Polycomb-dependent and -independent mechanisms to directly repress the abaxial determinants MIR166A, YABBY5, and AUXIN RESPONSE FACTOR3 (ARF3), as well as a nonrepressive mechanism in the regulation of the adaxial determinant TAS3A. These regulatory interactions, together with data from prior studies, lead to a model in which the sequential polarization of determinants, including AS1-AS2, explains the establishment and maintenance of adaxial-abaxial leaf polarity. Moreover, our analyses show that the shared repression of ARF3 by the AS and trans-acting small interfering RNA (ta-siRNA) pathways intersects with additional AS1-AS2 targets to affect multiple nodes in leaf development, impacting polarity as well as leaf complexity. These data illustrate the surprisingly multifaceted contribution of AS1-AS2 to leaf development showing that, in conjunction with the ta-siRNA pathway, AS1-AS2 keeps the Arabidopsis leaf both flat and simple.

Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.

The late steps of plant nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC-containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation. Our data indicate that the cysteine-histidine-rich (CH) and helicase domains of UPF1 are only essential for the early steps of NMD, whereas the heavily phosphorylated N- and C-terminal regions play a redundant but essential role in the target transcript degradation steps of NMD. We also show that both the N- and the C-terminal regions of SMG7 are essential for NMD. The N terminus contains a phosphoserine-binding domain that is required for the early steps of NMD, whereas the C terminus is required to trigger the degradation of NMD target transcripts. Moreover, SMG7 is a P-body component that can also remobilize UPF1 from the cytoplasm into processing bodies (P bodies). We propose that the N- and C-terminal phosphorylated regions of UPF1 recruit SMG7 to the functional NMD complex, and then SMG7 transports the PTC-containing transcripts into P bodies for degradation.

Boundary Formation through a Direct Threshold-Based Readout of Mobile Small RNA Gradients.

Small RNAs have emerged as a new class of mobile signals. Here, we investigate their mechanism of action and show that mobile small RNAs generate sharply defined domains of target gene expression through an intrinsic and direct threshold-based readout of their mobility gradients. This readout is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. Besides patterning their immediate targets, the readouts of opposing small RNA gradients enable specification of robust, uniformly positioned developmental boundaries. These patterning properties of small RNAs are reminiscent of those of animal morphogens. However, their mode of action and the intrinsic nature of their gradients distinguish mobile small RNAs from classical morphogens and present a unique direct mechanism through which to relay positional information. Mobile small RNAs and their targets thus emerge as highly portable, evolutionarily tractable regulatory modules through which to create pattern.

Developmental patterning by gradients of mobile small RNAs.

Development of multicellular organisms depends on intercellular communication via mobile signals that provide positional information to coordinate cell fate decisions. In addition to peptide ligands, transcription factors, and hormones, plants use small RNAs as positional instructive signals. The unique patterning properties of small RNA gradients resulting from regulated mobility suggest conceptual similarities to the function of animal morphogens, and provide robustness and precision to the formation of cell fate boundaries. While common principles may underlie the formation, stability, and interpretation of both plant small RNA and animal morphogen gradients, the unique nature of small RNAs with respect to their biogenesis and target regulation imply important differences as well. In this review, we discuss the patterning properties of mobile small RNAs and highlight recent studies that have advanced our understanding of how small RNAs move, and how the graded accumulation that underlies their patterning activity could be created, maintained, and interpreted.