Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2alpha phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.

Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

The Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.

HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.

Characteristics of transformed and tumor cells include increased levels of protein synthesis and elevated expression of RNA polymerase (pol) III products, such as tRNAs and 5S rRNA. However, whether deregulated pol III transcription contributes to transformation has been unclear. Generating cell lines expressing an inducible pol III-specific transcription factor, Brf1, allowed us to raise tRNA and 5S rRNA levels specifically. Brf1 induction caused an increase in cell proliferation and oncogenic transformation, whereas depletion of Brf1 impeded transformation. Among the gene products induced by Brf1 is the tRNA(iMet) that initiates polypeptide synthesis. Overexpression of tRNA(iMet) is sufficient to stimulate cell proliferation and allow immortalized fibroblasts to form foci in culture and tumors in mice. The data indicate that elevated tRNA synthesis can promote cellular transformation.

A suppressor gene was identified, which in high copy number rescues a temperature-sensitive mutation in yeast TATA-binding protein (TBP). Suppression was allele specific because the suppressor did not rescue the temperature-sensitive phenotype of another TBP mutant. This suppressor gene encodes a 596-amino-acid protein of which the amino-terminal half is homologous to the Pol II-specific factor TFIIB. Disruption of this gene, termed BRF1, showed it to be essential for growth of yeast. Deletion of sequences at either the amino or carboxyl terminus of BRF1 gave both temperature- and cold-sensitive phenotypes. These temperature- and cold-sensitive strains were used to prepare extracts deficient in BRF1 activity and were tested for transcriptional activity by RNA polymerases I, II, and III in vitro. BRF1-deficient extracts are defective in Pol III transcription and can be reconstituted for Pol III transcription by the addition of recombinant BRF1. Western analysis shows that BRF1 is present in TFIIIB but not the TFIIIC fraction, suggesting that it is a component of TFIIIB. We propose that BRF1 plays a role in Pol III initiation analogous to the role played by TFIIB for Pol II in its interaction with TBP and polymerase. The identification of a Pol III-specific TFIIB-like factor extends the previously noted similarity of transcriptional initiation by the three nuclear polymerases.

We report genome-wide analyses that establish Maf1 as a general and direct repressor of yeast RNA polymerase (Pol) III transcription. Chromatin immunoprecipitation (ChIP) coupled to microarray hybridization experiments showed an increased association of Maf1 to Pol III-transcribed genes under repressing condition (rapamycin treatment) correlated with a dissociation of Brf1 and Pol III. Maf1 can exist in various phosphorylation states and interacts with Pol III in a dephosphorylated state. The largest subunit of Pol III, C160, was identified as a target of Maf1. Under repressing conditions, Maf1 is dephosphorylated and accumulates in the nucleus, and Pol III-Maf1 interaction increases. Mutations in protein phosphatase type 2A (PP2A) catalytic subunit-encoding genes prevented rapamycin-induced Maf1 dephosphorylation, its nuclear accumulation, and repression of Pol III transcription. The results indicate that Pol III transcription can be globally and rapidly downregulated via dephosphorylation and relocation of a general negative cofactor.

RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation.

Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3' untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE-containing mRNA (ARE-mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14-3-3. In vivo and in vitro data support a model where PKB causes ARE-mRNA stabilization by inactivating BRF1 through binding to 14-3-3.

The tumor suppressor p53 is a transcription factor that controls cellular growth and proliferation. p53 targets include RNA polymerase (pol) III-dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA-binding protein (TBP)-containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP-p53 interaction is of functional importance for regulating RNA pol III-transcribed genes. Unlike RNA pol II-dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB.

Cells undergo various adaptive measures in response to stress. Among these are specific changes in the posttranscriptional regulation of various genes. In particular, the turnover of mRNA is modified to either increase or decrease the abundance of certain target messages. Some of the best-studied mRNAs that are affected by stress are those that contain adenine/uridine-rich elements (AREs) in their 3'-untranslated regions. ARE-containing mRNAs are involved in many important cellular processes and are normally labile, but in response to stress they are differentially regulated through the concerted efforts of ARE-binding proteins (AUBPs) such as HuR, AUF1, tristetraprolin, BRF1, and KSRP, along with microRNA-mediated effects. Additionally, the fate of ARE-containing mRNAs is modified by inducing their localization to stress granules or mRNA processing bodies. Coordination of these various mechanisms controls the turnover of ARE-containing mRNAs, and thereby enables proper responses to cellular stress. In this review, we discuss how AUBPs regulate their target mRNAs in response to stress, along with the involvement of cytoplasmic granules in this process.

The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.

In Saccharomyces cerevisiae, Maf1 is essential for mediating the repression of transcription by RNA polymerase (pol) III in response to diverse cellular conditions. These conditions activate distinct signaling pathways that converge at or above Maf1. Thus, Maf1-dependent repression is thought to involve a common set of downstream inhibitory effects on the pol III machinery. Here we provide support for this view and define two steps in Maf1-dependent transcriptional repression. We show that chlorpromazine (CPZ)-induced repression of pol III transcription is achieved by inhibiting de novo assembly of transcription factor (TF) IIIB onto DNA as well as the recruitment of pol III to preassembled TFIIIB.DNA complexes. Additionally Brf1 was identified as a target of repression in extracts of CPZ-treated cells. Maf1-Brf1 and Maf1-pol III interactions were implicated in the inhibition of TFIIIB.DNA complex assembly and polymerase recruitment by recombinant Maf1. Co-immunoprecipitation experiments confirmed these interactions in yeast extracts and demonstrated that Maf1 does not differentially sequester Brf1 or pol III under repressing conditions. The results suggest that Maf1 functions by a non-stoichiometric mechanism to repress pol III transcription.

Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNA(Leu) genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.

The TATA-binding proteins (TBP) from both human and Drosophila have been shown to exist in various distinct multiprotein complexes that are required, respectively, for transcription by all three RNA polymerases. In contrast, in vitro biochemical analyses have suggested that yeast TBP exists as a monomeric 27-kDa protein free in solution. We have examined the oligomerization state of yeast TBP and report here that yeast TBP, like human and Drosophila TBPs, is also stably associated with other proteins in vitro. Using anti-TBP antibodies we have immunopurified yeast TBP and associated factors (TBP-associated factors or TAFs). When this fraction was analyzed by SDS-polyacrylamide gel electrophoresis, polypeptides of approximate relative molecular size ranging from 170 to 60 kDa are prominently represented. Immunoblot analysis revealed that one of these TAFs, TAF70, corresponds to BRF1/TDS4/PCF4, a subunit of transcription factor (TF) IIIB. Furthermore, this highly purified TAF fraction can reconstitute polymerase III transcription when supplemented with purified RNA polymerase III and TFIIIC. Our data indicate that our TAF fraction contains TFIIIB transcription factor activity and that all the subunits of yeast TFIIIB are stably complexed with TBP.

RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNA(Leu) genes in cells decreases when ERK is inactivated. Furthermore, mutation of an ERK docking domain or phosphoacceptor site in BRF1 prevents serum induction of pol III transcription. These data identify a novel target for ERK, and suggest that its ability to stimulate biosynthetic capacity and growth involves direct transcriptional activation of tRNA and 5S rRNA genes.

BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBalpha(-/-) cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

Mitosis involves a generalized repression of gene expression. In the case of RNA polymerase III transcription, this is due to phosphorylation-mediated inactivation of TFIIIB, an essential complex comprising the TATA-binding protein TBP and the TAF subunits Brf1 and Bdp1. In HeLa cells, this repression is mediated by a mitotic kinase other than cdc2-cyclin B and is antagonized by protein phosphatase 2A. Brf1 is hyperphosphorylated in metaphase-arrested cells, but remains associated with promoters in condensed chromosomes, along with TBP. In contrast, Bdp1 is selectively released. Repression can be reversed by raising the concentration of Brf1 or Bdp1. The data support a model in which hyperphosphorylation disrupts TFIIIB during mitosis, compromising its ability to support transcription.

Transcription factor IIIB (TFIIIB) is directly involved in transcription initiation by RNA polymerase III in eukaryotes. Yeast contain a single TFIIIB activity that is comprised of the TATA-binding protein (TBP), TFIIB-related factor 1 (BRF1), and TFIIIB", whereas two distinct TFIIIB activities, TFIIIB-alpha and TFIIIB-beta, have been described in human cells. Human TFIIIB-beta is required for transcription of genes with internal promoter elements, and contains TBP, a TFIIIB" homologue (TFIIIB150), and a BRF1 homologue (TFIIIB90), whereas TFIIIB-alpha is required for transcription of genes with promoter elements upstream of the initiation site. Here we describe the identification, cloning, and characterization of TFIIIB50, a novel homologue of TFIIB and TFIIIB90. TFIIIB50 and tightly associated factors, along with TBP and TFIIIB150, reconstitute human TFIIIB-alpha activity. Thus, higher eukaryotes, in contrast to the yeast Saccharomyces cerevisiae, have evolved two distinct TFIIB-related factors that mediate promoter selectivity by RNA polymerase III.

Regulated mRNA decay is a highly important process for the tight control of gene expression. Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3' untranslated regions that direct rapid mRNA decay by interaction with decay-promoting ARE-binding proteins (ARE-BPs). The decay of ARE-containing mRNAs is regulated by signaling pathways that are believed to directly target ARE-BPs. Here, we show that BRF1 involved in ARE-mediated mRNA decay (AMD) is phosphorylated by MAPK-activated protein kinase 2 (MK2). In vitro kinase assays using different BRF1 fragments suggest that MK2 phosphorylates BRF1 at four distinct sites, S54, S92, S203, and an unidentified site at the C terminus. Coexpression of an active form of MK2 inhibits ARE mRNA decay activity of BRF1. MK2-mediated inhibition of BRF1 requires phosphorylation at S54, S92, and S203. Phosphorylation of BRF1 by MK2 does not appear to alter its ability to interact with AREs or to associate with mRNA decay enzymes. Thus, MK2 inhibits BRF1-dependent AMD through direct phosphorylation. Although the mechanism underlying this inhibition is still unclear, it appears to target BRF1-dependent AMD at a level downstream from RNA binding and the recruitment of mRNA decay enzymes.