K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis.

Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.

3' Rapid Amplification of cDNA Ends (3' RACE) Using Arabidopsis Samples.

Production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and various RNA processing activities, including splicing, 5' capping, and 3' cleavage and polyadenylation (Bentley, 2014). Accurate mapping of RNA ends provides a valuable tool to assess transcriptional and post-transcriptional events giving rise to different gene transcripts. The abundance of such transcripts most likely depends on exogenous and developmental cues, or mutations. In the reference plant Arabidopsis, perturbation of the HUA-PEP post-transcriptional regulatory factors (Rodríguez-Cazorla et al., 2015) leads to the accumulation of aberrant transcripts of the key floral homeotic gene AGAMOUS (AG) (Yanofsky et al., 1990) that retain intronic sequence. It was determined by 3' RACE reactions that such erroneous transcripts correspond to premature processing and polyadenylation events taking place at the AG intron region. Here we describe a protocol that is suitable for analysis of relatively abundant transcripts and also for detecting aberrant RNA species that are likely prone to rapid turnover. Likewise, the method, here adapted to Arabidopsis reproductive tissues, can be applied to characterize RNA species from other organs (leaf, root) and/or other plant species. We provide a detailed protocol of our 3' RACE procedure comprising four major parts: Total RNA extraction, RNA amount determination and quality control, the RACE procedure itself, and isolation of the resulting RACE products for cloning and sequencing.

Antagonistic interactions between Arabidopsis K-homology domain genes uncover PEPPER as a positive regulator of the central floral repressor FLOWERING LOCUS C.

Plant floral transition is a major developmental switch regulated by an integrated network of pathways. Arabidopsis FLOWERING LOCUS K (FLK), a protein with three KH RNA-binding domains, operates in the autonomous flowering-promotive pathway by decreasing the transcript levels of the key flowering repressor FLOWERING LOCUS C (FLC). Here we report that PEPPER (PEP), an FLK paralog previously shown to affect vegetative and pistil development, antagonizes FLK by positively regulating FLC. Lack of PEP function rescues the flk late-flowering phenotype with a concomitant decrease in FLC RNA levels. Loss of HUA2, another FLC activator encoding an RNA-binding protein, further rescues flk, being flk hua2 pep triple mutants virtually wild-type regarding flowering time. Consistently, PEP overexpression determines high levels of FLC transcripts and flowering delay. Genetic and molecular analyses indicate that FLK and PEP act independently of FCA, another important FLC repressor in the autonomous pathway. In addition, we present data suggesting that PEP may affect FLC expression at both transcriptional and post-transcriptional levels. Overall, our results uncover PEP as a new factor for FLC upregulation, underscoring the importance of RNA-binding activities during developmental timing of flowering.

Intraphylum diversity and complex evolution of cyanobacterial aminoacyl-tRNA synthetases.

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.

Regulated expression of glutamyl-tRNA synthetase is directed by a mobile genetic element in the cyanobacterium Tolypothrix sp. PCC 7601.

The genome of Tolypothrix sp. PCC 7601 carries two copies of a novel insertion sequence, ISTosp1. One of the two copies is located upstream of the gene encoding glutamyl-tRNA synthetase, an enzyme playing a key role in protein and pigment synthesis. The tnpA gene of the IS element and gltX were co-transcribed and their expression was transiently upregulated upon retrieval of the ammonium source irrespective of whether nitrate or no nitrogen source were available. The second copy is also transcribed and shows a similar regulatory pattern. Structural elements of the promoter (-10 and -35 sequences) directing the expression of the tnpA-gltX operon have been localized within the IS. Regulatory sequences involving the NtcA transcription factor in the control of tnpA-gltX expression were found both within and in sequences upstream of the insertion element. The expression of gltX in a closely related cyanobacterium, Nostoc sp. PCC 7120, which lacks the insertion upstream of gltX, decreased upon ammonium retrieval, a regulatory pattern that markedly differs from that observed in Tolypothrix sp. PCC 7601. ISTosp1 constitutes a good example of how cells can make use of a transposable element to evolve an original regulatory mechanism.