The ATXN2 Orthologs CID3 and CID4, Act Redundantly to In-Fluence Developmental Pathways throughout the Life Cycle of Arabidopsis thaliana.

RNA-binding proteins (RBPs) are key elements involved in post-transcriptional regulation. Ataxin-2 (ATXN2) is an evolutionarily conserved RBP protein, whose function has been studied in several model organisms, from Saccharomyces cerevisiae to the Homo sapiens. ATXN2 interacts with poly(A) binding proteins (PABP) and binds to specific sequences at the 3'UTR of target mRNAs to stabilize them. CTC-Interacting Domain3 (CID3) and CID4 are two ATXN2 orthologs present in plant genomes whose function is unknown. In the present study, phenotypical and transcriptome profiling were used to examine the role of CID3 and CID4 in Arabidopsis thaliana. We found that they act redundantly to influence pathways throughout the life cycle. cid3cid4 double mutant showed a delay in flowering time and a reduced rosette size. Transcriptome profiling revealed that key factors that promote floral transition and floral meristem identity were downregulated in cid3cid4 whereas the flowering repressor FLOWERING LOCUS C (FLC) was upregulated. Expression of key factors in the photoperiodic regulation of flowering and circadian clock pathways, were also altered in cid3cid4, as well as the expression of several transcription factors and miRNAs encoding genes involved in leaf growth dynamics. These findings reveal that ATXN2 orthologs may have a role in developmental pathways throughout the life cycle of plants.

Molecular basis for neofunctionalization of duplicated E3 ubiquitin ligases underlying adaptation to drought tolerance in Arabidopsis thaliana.

Multigene families in plants expanded from ancestral genes via gene duplication mechanisms constitute a significant fraction of the coding genome. Although most duplicated genes are lost over time, many are retained in the genome. Clusters of tandemly arrayed genes are commonly found in the plant genome where they can promote expansion of gene families. In the present study, promoter fusion to the GUS reporter gene was used to examine the promoter architecture of duplicated E3 ligase genes that are part of group C in the Arabidopsis thaliana ATL family. Acquisition of gene expression by AtATL78, possibly generated from defective AtATL81 expression, is described. AtATL78 expression was purportedly enhanced by insertion of a TATA box within the core promoter region after a short tandem duplication that occurred during evolution of Brassicaceae lineages. This gene is associated with an adaptation to drought tolerance of A. thaliana. These findings also suggest duplicated genes could serve as a reservoir of tacit genetic information, and expression of these duplicated genes is activated upon acquisition of core promoter sequences. Remarkably, drought transcriptome profiling in response to rehydration suggests that ATL78-dependent gene expression predominantly affects genes with root-specific activities.

CTLs, a new class of RING-H2 ubiquitin ligases uncovered by YEELL, a motif close to the RING domain that is present across eukaryotes.

RING ubiquitin E3 ligases enclose a RING domain for ubiquitin ligase activity and associated domains and/or conserved motifs outside the RING domain that collectively facilitate their classification and usually reveal some of key information related to mechanism of action. Here we describe a new family of E3 ligases that encodes a RING-H2 domain related in sequence to the ATL and BTL RING-H2 domains. This family, named CTL, encodes a motif designed as YEELL that expands 21 amino acids next to the RING-H2 domain that is present across most eukaryotic lineages. E3 ubiquitin ligase BIG BROTHER is a plant CTL that regulates organ size, and SUMO-targeted ubiquitin E3 ligase RNF111/ARKADIA is a vertebrate CTL. Basal animal and vertebrate, as well as fungi species, encode a single CTL gene that constraints the number of paralogs observed in vertebrates. Conversely, as previously described in ATL and BTL families in plants, CTL genes range from a single copy in green algae and 3 to 5 copies in basal species to 9 to 35 copies in angiosperms. Our analysis describes key structural features of a novel family of E3 ubiquitin ligases as an integral component of the set of core eukaryotic genes.

Expansion and diversification of BTL ring-H2 ubiquitin ligases in angiosperms: putative Rabring7/BCA2 orthologs.

RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes.

Diversity in the architecture of ATLs, a family of plant ubiquitin-ligases, leads to recognition and targeting of substrates in different cellular environments.

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20-28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress.

Genetic interactions of a putative Arabidopsis thaliana ubiquitin-ligase with components of the Saccharomyces cerevisiae ubiquitination machinery.

The feasibility of using the Saccharomyces cerevisiae genetic tools to get insights into the function of a plant-specific ubiquitin-ligase was examined. ATL2 is a potential ubiquitin-ligase of the RING-H2 type that was originally isolated as a conditionally toxic Arabidopsis cDNA when overexpressed in yeast. ATL2 is a member of an Arabidopsis family that comprises 80 proteins. After testing cDNAs from 25 ATL members for toxicity we found that in addition to ATL2 only ATL63 was toxic, suggesting specific interactions of each one of these two ATLs in yeast. We seek to identify suppressors of the ATL2 toxicity in yeast and we found that toxicity was suppressed by knock-out mutations on different components of the ubiquitination pathway. Suppression was achieved in four deubiquitinating enzyme mutants and in one ubiquitin-conjugating enzyme mutant. A model is proposed in which Ubc4 and ATL2 act together to target for degradation one or more essential yeast proteins, Doa4/Ubp4, Ubp6 and Ubp14 have a role in disassembling ubiquitin chains on the target proteins and Ubp15 protects ATL2 from auto-ubiquitination. We presuppose that our approach can be further utilized to analyze the function of this distinctive class of ubiquitin-ligases in yeast as well as in Arabidopsis.

ETHYLENE-INSENSITIVE5 encodes a 5'-->3' exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2.

Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence of ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F-box proteins, EBF1 and EBF2. Here we report the identification of ETHYLENE-INSENSITIVE5 as the 5'-->3' exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response.

Four distinct classes of proteins as interaction partners of the PABC domain of Arabidopsis thaliana Poly(A)-binding proteins.

Poly(A)-binding proteins (PABPs) play an important role in the regulation of translation and the control of mRNA stability in eukaryotes, and their functions are known to be essential in many organisms. PABPs contain a highly conserved C-terminal segment termed the PABC domain. The PABC domain from human PABP interacts with the proteins PAIP1, PAIP2 and RF3 via its PAM2 motifs. These interactions are important for modulating translation. Arabidopsis has eight PABPs, an unexpectedly large number in comparison to other eukaryotes whose genomes have been sequenced. Six of the Arabidopsis PABPs contain the conserved PABC domain. In this work, we have identified PABC-interacting proteins in Arabidopsis. Two proteins, which we named CID1 and CID7, were initially isolated in a two-hybrid screen, and eleven more were predicted to be present in the Arabidopsis proteome and eleven in the rice proteome. Among the 24 PAM2-containing proteins in this set, we observed a diversity of modules of intriguing function, ranging from acidic regions similar to the PAM1 motif found in human PAIP1 and PAIP2, to domains such as the small MutS-related domain, the Lsm domains of Ataxin-2, and RNA recognition motifs (RRMs). We suggest that the large number of PABPs and PAM2-containing proteins may have evolved to provide plants with greater flexibility in modulating the metabolism of specific transcripts. We also found that two PABP genes, PAB2 (ubiquitously expressed) and PAB5 (expressed in reproductive tissues), are essential for viability, suggesting that each has a vital and specific function.

Genome-wide insertional mutagenesis of Arabidopsis thaliana.

Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.