Effect of a natural mutation in the 5 ` untranslated region on the translational control of p53 mRNA

Tumor-suppressor protein p53, the `guardian of the genome', is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, Delta N-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5' untranslated region (5' UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5' UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5' UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5' UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2-M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5' UTR. Taken together, the results demonstrate for the first time that SNP in the 5' UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene.

SRF Phosphorylation by Glycogen Synthase Kinase-3 Promotes Axon Growth in Hippocampal Neurons

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

Endoplasmic Reticulum Stress-Mediated Activation of p38 MAPK, Caspase-2 and Caspase-8 Leads to Abrin-Induced Apoptosis

Abrin from Abrus precatorius plant is a potent protein synthesis inhibitor and induces apoptosis in cells. However, the relationship between inhibition of protein synthesis and apoptosis is not well understood. Inhibition of protein synthesis by abrin can lead to accumulation of unfolded protein in the endoplasmic reticulum causing ER stress. The observation of phosphorylation of eukaryotic initiation factor 2 alpha and upregulation of CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. Moreover, abrin-induced apoptosis was found to be dependent on p38 MAPK but not JNK. We also observed that abrin induced the activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery.

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis

Background: DNA-binding protein from starved cells (Dps) are nano-compartments that can oxidize and store iron rendering protection from free radicals. Results: A histidine-aspartate ionic cluster in mycobaterial Dps2 modulates the rate of iron entry and exit in these proteins. Conclusion: Substitutions that disrupt the cluster interface alter the iron uptake/release properties with localized structural changes. Significance: Identifying important gating residues can help in designing nano-delivery vehicles. Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 for the various mutants to compare structural alterations vis a vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

Titania nanotube-modified screen printed carbon electrodes enhance the sensitivity in the electrochemical detection of proteins

The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (C-protein ca 1 ng/mu l). Reproducible measurements on protein samples at this concentration (I-p,I-a of 80 +/- 1.2 mu A) could be achieved using a sample volume of ca 30 mu l. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments. (C) 2014 Elsevier B.V. All rights reserved.

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP-promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion

Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface alpha 5 beta 1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 x 10(8)) and Tomlinson J (Library size 1.37 x 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

Reversible induction of translational isoforms of p53 in glucose deprivation

Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Delta 40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tract-binding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Delta 40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Delta 40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation.

Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C-terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium ΔaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4 log10 colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium ΔaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to ΔspaS or ΔssaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the ΔaroA mutant. Expression in the ΔaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry. © 2009 Elsevier Ltd. All rights reserved.

Association analyses of DNA polymorphisms in bovine SREBP-1, LXRα,FADS1 genes with fatty acid composition in Canadian commercial crossbred beef steers

Two previously reported DNA polymorphisms of sterol regulatory element binding transcription factor 1 (SREBP1) and liver X receptor alpha (LXRα) and two DNA polymorphisms of fatty acid desaturase 1 (FADS1) were evaluated for associations with fatty acids in brisket adipose tissue of Canadian cross-bred beef steers. The polymorphism of 84 bp insert/deletion in intron 5 of SREBP1 was significantly associated with the concentration of 9c C17:1 (P=0.013). The G>A single nucleotide polymorphism (SNP) in the exon 4 of LXRα gene was associated with the concentration of 9c, 11t C18:2 (P=0.04), sum of conjugated linoleic acids (CLA) (P=0.025) and 11c C20:1(P=0.042). Two DNA polymorphisms in the promoter region of FADS1, deletion/insertion of ->GTG in rs133053720 and SNP A>G in rs42187276, were significantly associated with concentrations of C17:0 iso, C17:0 ai, total branched chain fatty acids (BFA), 12t C18:1, 13t/14t C18:1, 15t C18:1, and 13c C18:1 (P<0.05). Further studies are needed to validate the associations and to delineate the roles of the gene polymorphisms in determining the fatty acid composition in beef tissues.

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Brf1 post-transcriptionally regulates pluripotency and differentiation responses downstream of Erk MAP Kinase

FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.