The tRNA m 1 A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m 1 A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m 1 A58 and consequential tRNA i Met instability. We used a genetic approach to identify the genes responsible for tRNA i Met degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNA i Met to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3-5 exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNA i The relatively unstable nature of messenger RNAs fueled the discovery of pathways that control the degradation of normal and abnormal mRNAs in the nucleus and cytoplasm (Hilleren and Parker 1999;Mitchell and Tollervey 2001;Wilusz et al. 2001;Maquat 2002;Moore 2002;Long and McNally 2003). Two general pathways of mRNA decay have been characterized in the yeast Saccharomyces cerevisiae, and homologs of most of the yeast proteins involved in mRNA turnover have been identified in metazoans. The first pathway initially requires shortening of the mRNA polyadenylate tail, followed by removal of the 5Ј cap structure (Wilusz et al. 2001), which leaves the body of the mRNA susceptible to 5Ј-3Ј exonucleolytic degradation by Xrn1p. The second pathway involves deadenylation of mRNAs and the 3Ј-5Ј degradation of the body of the mRNA by the exosome (Jacobs et al. 1998;Burkard and Butler 2000;van Hoof et al. 2000b;van Hoof and Parker 2002;Mitchell and Tollervey 2003).The exosome is a multisubunit complex of proteins with multiple functions in the processing, degradation, and retention of stable and unstable RNAs in the nucleus and cytoplasm. The cytoplasmic exosome directly interacts with Ski7p (Araki et al. 2001) and recruits the Ski2p, Ski3p, and Ski8p complex to the 3Ј end of a deadenylated mRNA (Brown et al. 2000) or an mRNA that is stalled on the ribosome because it lacks a stop codon (Jacobs et al. 1998;van Hoof et al. 2000b), and in turn each is degraded in a 3Ј-to-5Ј direction. In the nucleus, the exosome has been implicated in elimination of by-products of rRNA processing (ETS sequence). The nuclear exosome possesses an exonuclease, Rrp6p, not found in the cytoplasmic form (Allmang et al. 1999b). A specialized function of Rrp6p and the nuclear exosome appears to be in retaining mRNAs incorrectly processed at their 3Ј ends at the site of transcription to prevent their release into the cytoplasm (Hilleren et al. 2001;Libri et al. 2002). Thus far, the exosome has not been implicated in the destruction of stable RNAs that are rendered unstable due to mutations or defects in processing.

Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.Eukaryotic activator proteins stimulate transcription by binding to their target genes and carrying out two general functions: (i) altering the locations or structures of nucleosomes and (ii) recruiting TATA-binding protein (TBP), other general transcription factors (GTFs), and RNA polymerase II (RNA PolII) to the promoter. Most activators carry out these functions indirectly by recruiting multisubunit complexes, collectively called coactivators (39,70,90). One class of coactivators uses ATP hydrolysis to displace nucleosomes and thereby expose or obscure protein binding sites in the promoter (91,124). Each of the nucleosome remodeling complexes of Saccharomyces cerevisiae, known as SWI/SNF, RSC, ISW1, and ISW2, contains a different subunit harboring the ATPase activity of the complex (reviewed in references 70 and 91). Although each has been implicated in transcriptional activation in vivo (5,38,51,85,123), only the nonessential SWI/SNF complex has been shown to interact physically with activators (93, 139) and be recruited to a promoter for nucleosome remodeling and transcriptional activation in vitro (45,96,139). Recruitment of the SWI/SNF complex by yeast activators has also been demonstrated in living yeast cells by chromatin immunoprecipitation (ChIP) assays (24, 126).Another class of coactivators alters chromatin structure by acetylation of lysines in the amino-terminal tails of histones. This modification destabilizes higher-order chromatin structure (116) and also may stimulate binding of other coactivator proteins containing a bromodomain (9,91,120,135). The SAGA complex is the best-characterized yeast coactivato...

The microbiome of MEEs from children with chronic otitis media differs according to specific clinical features, such as mucin content, age and presence of hearing loss. These associations provide novel pathophysiologic insights across the spectrum of otitis media progression.

BackgroundChronic Otitis Media with effusion (COME) is characterized by persistent middle ear effusions that are in most cases highly viscous, but some patients present with serous fluid. This study aimed at comprehensively characterizing the macromolecular composition of mucoid vs. serous MEEs.MethodsMEEs from patients with COME were analyzed for proteins by Mass Spectrometry (MS) and western blot techniques, total DNA quantity, bacterial DNA (16S sequencing) and cytokine content. Proteomics datasets were studied in Ingenuity pathway analysis (IPA).ResultsMucoid samples showed a global tendency of increased pro- inflammatory mediators. IL1-β and IL-10 were significantly more abundant in serous samples (p<0.01). Mucoid samples had higher DNA quantity (p=0.04), more likely to be positive in MUC5B protein (p=0.008) and higher peptide counts (12786 vs 2225), as well as an overall larger number of identified proteins (331 vs 177), compared to serous. IPA found the mucoid sample dataset to be related to immune cell function and epithelial remodeling whereas the serous sample dataset showed acute responses and blood related proteins. Interestingly, serous samples showed more bacterial DNA than mucoid ones, with less bacterial genera variability.ConclusionThis study demonstrates divergent immune responses in children with COME by effusion quality.

To our knowledge, this the first report demonstrating the presence of exosomes transporting miRNAs in MEEs. These findings open a broad and novel area of research in OM pathophysiology as driven by miRNA cell communication.