A new emerging monocot model species

Brachypodium distachyon, a newly sequenced monocot representative model species shows promise in the area of plant science research. Brachypodium is alluring as a model species because it is closely related to agriculturally relevant crop species such as corn, rice, and switchgrass. The new model species was sequenced no more than one year ago, and scientific researchers have already begun digging through the genome, uncovering nature's secrets. Much knowledge can be transferred from the Arabidopsis community to this new model species. Several ideas have already carried over, such as an online genomics database with annotations and novel bioinformatics assistance such as the BLAST tool. We (the holt lab) recently published a genomics survey paper in the Public Library of Science (PLoS one) pertaining to identification and characterization of the transcription factor families that we work on in Arabidopsis. We used the BLAST tool to match protein sequences from the Arabidopsis genome against the Brachypodium genome to find orthologous genes across species. We identified several an equal number of A subunits, and an expanded number of B and C subunits in Brachypodium. What set this paper apart from other genomics surveys was the genetic characterization. We cloned a predicted Brachypodium orthologue and inserted it into an Arabidopsis mutant background. The gene was in fact an orthologue due to its ability to rescue the mutant phenotype in a different species of plant. This shows the strong influence of evolution and the importance of NF-Y transcription factors in the plant kingdom. Monocot and dicot families experienced millions of years of evolutionary diversity, all the while retaining relatively similar genetics. If that is not evidence of a common ancestor, I do not know what is.

splice-athon

I was looking through pubmed the other day for papers pertaining to transcriptomics, alternatively referred to as RNA-sequencing. This was an assignment for the other course I am taking this semester - Transcriptomics, which is taught by an E. coli-ologist at SRTC. The assignment was straightforward: find a paper in your field pertaining to RNA-seq. I ended up choosing a paper that evaluated the downstream activity of a type II protein arginine methyl transferase (PRMT). These specialized proteins add methyl groups to arginine residues to other proteins, primarily RNA-binding proteins. This particular PRMT had been initially assessed in humans before researchers in my field utilized bioinformatics to find the homologue. The Arabidopsis homologue of PRMT5 was shown to a be a precursor for proper pre-mRNA splicing. The experiments were elegantly designed for the level of complexity that the research possessed, which made this a special read. They mutated AtPRMT5 (inserted ~10kb DNA junk in coding region of gene), extracted total RNA and used this template, along with wild-type (WT), for SOLiD RNA-seq. The results were profound. The transcriptome landscape in the mutant background was quite divergent from that of the WT reads. The scientist looked for mis-spliced transcripts, in other words, transcripts that contained activity through introns. If the mRNA was being properly spliced, there would not be introns present, or there would be no activity through intronic regions. They identified ~14 genes that were mis-spliced. the most important, and phenotypically quantifiable of their findings was the misregulated mRNA splicing of the floral regulator FLK (FLOWERING LOCUS KH DOMAIN) which is a negative regulator of FLC (FLOWERING LOCUS C) which directly down-regulates key floral initiation genes. FLK was shown to be mis-spliced in the prmt5 background, but contained a normal transcript in the WT background. This was confirmed by RNA blot , which showed one band in the WT background, where the mutant background contained splice variants of FLK, consistent with the RNA-seq findings. Next, they examined FLC expression in WT and prmt5 backgrounds. FLC was weakly expressed in the WT background, and strongly expressed in the mutant background. This finding suggests that the mis-splicing of FLK is unable to negatively regulate the expression of FLC, which must be down-regulated to properly transition to the reproductive state. They then examined flowering time of WT and mutant plants. prmt5 plants flowered twice as slow as WT plants due to the misregulation of the floral repressor, FLC. I thought it was both amazing and elegant that such careful experimental design could tie the smallest genetic discrepancy to such a grossly recognizable phenotype. That is all.

Biochemistry of the Nuclear Factor Y

Before I get started, I would like to address the serious problem that I have with the geniuses of the world. Occasionally while reading the literature in my field, I begin to feel a strong wave of hopelessness wash over me. However, I was told by an elder at some point in my academic career that, "If you've made it this far, you're smart enough." Whenever I feel overwhelmed, I sit back, reflect, and run this quote through my mind. Back to the geniuses. An Italian molecular biologist named Roberto Mantovani is to thank for pioneering NF-Y research starting in the early 1990's and continuing through 2011 with his latest publication in PLoS.
In mammalian and plant systems, there are three NF-Y families, or subunits: NF-YA, NF-YB, and NF-YC. Mammalian biochemistry has revealed that the three subunits must form a trimer in order to properly bind DNA at the pentameric sequence CCAAT,  referred to as the CAT-box. Mammalian work has also shown that NF-Y posses compartment specificity. The working model is that NF-YB proteins and NF-YC proteins are localized primarily to the cytoplasm, until the B and C subunits dimerize. NF-YB "piggy-backs" on the NF-YC subunit, eventually penetrating the nuclear envelope. Once the B/C dimer has accessed the nucleus, the NF-YA/B/C trimer can bind sequence specific nucleotides in the promoters of genes. It is understood that the NF-Y trimer mimics an inverted triangle, with the A subunit occupying the apex. This apex, or A subunit is the only protein to physically contact the pentameric CAT-box. It is assumed that the B and C subunits grasp nucleotides flanking the CAT-box. This is important for a number of reasons. Nucleotides outside of the consensus sequence of ggCCAATct are not conserved, thus must be variable. This could explain NF-YB/C's loss of homology outside of the "conserved domain", which contains NF-YA/B/C binding domains and a DNA binding domain. Alternatively,  NF-YA subunits possess a conserved domain with almost twice the level of conservation as the B and C subunits.

cloning disaster

I strolled into the lab as eager as one could possibly be this morning. Let me rephrase that: as eager as any scientist could be. I walked directly to the 37 degree centigrade incubator to remove the two E. coli plates that I assumed harbored my recombinant plasmids. I performed several rounds of colony PCR in hopes of amplifying the unique target sequence that I was after, however, this resulted in nothing more than a fluorescent-less agarose gel. These are a pair of N-terminally truncated clones that I need in order to move forward with my research. I guess I'll tell you a little about my research now, since I've gotten the venting out of the way. 

I work on a family of specialized proteins called the Nuclear Factor Y (hereafter referred to as NF-Y). NF-Y are a heterotrimeric transcriptional complex that regulate downstream gene expression. These proteins were originally identified in yeast, and are called Heme Activator Proteins, or simply HAPs, in less advanced eukaryotic systems. These proteins require Iron ions in order to function, similar to other enzymes, namely polymerases, that require magnesium ions. Yeast contain one copy of each of the 3-4 HAP or NF-Y proteins. Conversely, all sequenced higher plants have been shown through bioinformatics to have experienced numerous evolutionary duplication events of NF-Y. For example, Arabidopsis thaliana possess 10 NF-YA, 13 NF-YB, and 13 NF-YC proteins. This evidence suggests that plants have duplicated and undergone slight mutations of these proteins for a broader range of developmental processes.