The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome.The adaptor protein CD2BP2,originally identified as a binding partner of the adhesion molecule CD2,is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine(GYF)domain to co-localize with spliceosomal proteins.So far,its function in vertebrates is unknown.Using conditional gene targeting in mice,we show that CD2BP2 is crucial for embryogenesis,leading to growth retardation,defects in vascularization,and premature death at embryonic day 10.5 when absent.Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins.At the molecular level,we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2.Given the strong expression of CD2BP2 in podocytes of the kidney,we use selective depletion of CD2BP2,in combination with next-generation sequencing,to monitor changes in exon usage of genes critical for podocyte functions,including VEGF and actin regulators.CD2BP2-depleted podocytes display foot process effacement,and cause proteinuria and ultimately lethal kidney failure in mice.Collectively,our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

Stem Cell Regulation by Arabidopsis WOX Genes

Gene amplification followed by functional diversification is a major force in evolution. A typical example of this is seen in the WUSCHEL-RELATED HOMEOBOX （WOX） gene family, named after the Arabidopsis stem cell regulator WUSCHEL. Here we analyze functional divergence in the WOX gene family. Members of the WUS clade, except the cambium stem cell regulator WOX4, can substitute for WUS function in shoot and floral stem cell maintenance to different degrees. Stem cell function of WUS requires a canonical WUS-box, essential for interaction with TPL/TPR co-repressors, whereas the repressive EAR domain is dispensable and the acidic domain seems only to be required for female fertility. In contrast to the WUS clade, members of the ancient WOX13 and the WOX9 clades cannot support stem cell maintenance. Although the homeodomains are interchangeable between WUS and WOX9 clade members, a WUS- compatible homeodomain together with canonical WUS-box is not sufficient for stem cell maintenance. Our results suggest that WOX function in shoot and floral meristems of Arabidopsis is restricted to the modern WUS clade, suggesting that stem cell control is a derived function. Yet undiscovered functional domains in addition to the homeodomain and the WUS-box are necessary for this function.

DICER-LIKE3 Activity in Physcomitrella patens DICER-LIKE4 Mutants Causes Severe Developmental Dysfunction and Sterility

Trans-acting small interfering RNAs （ta-siRNAs） are plant-specific siRNAs released from TAS precursor tran- scripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased process- ing. Like microRNAs （miRNAs）, ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens （PpDCL4） is essential for the biogenesis of 21 nucleotide （nt） ta-siRNAs. In APpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. APpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in APpDCL3/APpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the APpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TA56. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci.

AtNSF regulates leaf serration by modulating intracellular trafficking of PIN1 in Arabidopsis thaliana

In eukaryotes,N-ethylmaleimide-sensitive factor(NSF)is a conserved AAA+ATPase and a key component of the membrane trafficking machinery that promotes the fusion of secretory vesicles with target membranes.Here,we demonstrate that the Arabidopsis thaliana genome contains a single copy of NSF,At NSF,which plays an essential role in the regulation of leaf serration.The At NSF knock-down mutant,atnsf-1,exhibited more serrations in the leaf margin.Moreover,polar localization of the PINFORMED1(PIN1)auxin efflux transporter was diffuse around the margins of atnsf-1 leaves and root growth was inhibited in the atnsf-1 mutant.More PIN1-GFP accumulated in the intracellular compartments of atnsf-1 plants,suggesting that At NSF is required for intracellular trafficking of PIN between the endosome and plasma membrane.Furthermore,the serration phenotype was suppressed in the atnsf-1 pin1-8 double mutant,suggesting that At NSF is required for PIN1-mediated polar auxin transport to regulate leaf serration.The CUPSHAPED COTYLEDON2(CUC2)transcription factor gene is up-regulated in atnsf-1 plants and the cuc2-3 single mutant exhibits smooth leaf margins,demonstrating that At NSF also functions in the CUC2 pathway.Our results reveal that At NSF regulates the PIN1-generated auxin maxima with a CUC2-mediated feedback loop to control leaf serration.