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Coordinating Expression of RNA Binding Proteins with Their mRNA Targets

ABSTRACT: Post-transcriptional regulation by RNA binding proteins (RBPs) plays prominent roles in a variety of biological processes. In this study, by analyzing the global regulatory relationship between RBPs and their target mRNAs in yeast, we discovered that most RBP genes are co-regulated with their target genes, but the RBPs tend to dampen expression variation among their target mRNAs. We further examined a well-studied RBP gene, PUF3, and found that the protein decreases the variation of its target mRNAs by differentially affecting their decay. Our results provided new insights into the functional importance of RBPs in coordinating their target genes expression. Both the PUF3 deletion strain and wild type BY4742 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) strain were cultured overnight in YPD media. 0.01 OD cells were transferred into fresh YPEG media until 1.0 OD. Total RNA was extracted by the standard Trizol protocol. mRNA was then purified using oligo-dT DynaBeads. cDNA sequencing library was constructed according to the protocol described by Wang et al. After sequencing, the reads were also mapped into S. cerevisiae genome by SOAP2 with no more than two mismatches. RPKM was used to represent the expression level of each gene.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Huifeng Jiang

PROVIDER: E-GEOD-55419 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Coordinating Expression of RNA Binding Proteins with Their mRNA Targets

Project description:Post-transcriptional regulation by RNA binding proteins (RBPs) plays prominent roles in a variety of biological processes. In this study, by analyzing the global regulatory relationship between RBPs and their target mRNAs in yeast, we discovered that most RBP genes are co-regulated with their target genes, but the RBPs tend to dampen expression variation among their target mRNAs. We further examined a well-studied RBP gene, PUF3, and found that the protein decreases the variation of its target mRNAs by differentially affecting their decay. Our results provided new insights into the functional importance of RBPs in coordinating their target genes expression.

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2011-07-30 | E-GEOD-26171 | biostudies-arrayexpress

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Yeast genomic expression patterns in response to zymolyase treatment

Project description:The evaluation of zymolyase treatment using yeast gene expression comparison analysis. The yeast BY4743 strain was used for this study. Yeast cells were treated with Zymolyase (final conc. 300 U/ml), and incubated at 37 degree for ten minutes. Digestion process was not provided for control sample. Total RNA was isolated with commercial kit (FastRNA Pro Red kit, Q-Biogene), and amplified RNA (aRNA) was synthesized with 3'IVT Express kit (Affymetrix). All samples were hybridized with Gene Chip (Yeast Genome 2.0 Array, Affymetrix), then each array chip was scanned by GeneChip Sanner 3000 (Affymetrix). Yeast were treated with zymolyase or left untreated. The only difference in conditions between the treated and untreated samples was the enzymatic digestion process. The conditioned samples were normalized with three replicates.

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Proteomic Analysis of Protein Methylation in the Yeast Saccharomyces cerevisiae

Project description:Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the SAM-auxotrophic Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 117 methylated proteins, containing 182 methylation events associated with 174 methylation sites. About 90% of these methylation events were previously unknown. Our results indicated, 1) over 6% of the yeast proteome are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys >> Arg > Asp > Glu ≈ Gln ≈ Asp > His > Cys, 3) the methylation state on nitrogen center is largely exclusive, and 4) the methylated proteins are located mainly in nucleus/ribosome associated with translation/transcription and DNA/RNA processing. Our dataset is the most comprehensive methylproteome known-to-date of all living organisms, and should significantly contribute to the field of protein methylation and related research.

2020-01-27 | PXD000606 | Pride

Comparative Dynamic Transcriptome Analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

Project description:Data was pre-processed array-wise using expresso (R/Bioconductor) with the RMA background correction method. We created our own probe annotation environment (cdf), which excludes probes in probesets that show cross-hybridization between Sc and Sp. 8708 annotated Sc probes and 13,317 annotated Sp probes out of a total of 120,855 probes showed cross-hybridization when a conservative intensity cut-off of 4.5 (log intensity values after preprocessing) was used. Cross-hybridizing probes were excluded from further analysis. This included 16 whole probe sets. Note that the standard GC-RMA method is not suitable for our purposes, since its bias model cannot handle bimodal intensity distributions, as caused by the simultaneous hybridization of Sc and Sp transcripts with global differences in RNA abundance. Labeling bias estimation and correction was done as described (Miller et al. 2011). Between-array normalization of arrays containing mixed Sc and Sp total RNA was done by proportional rescaling, such that the median Sp gene expression level was 1. Accordingly, between-array normalization of arrays containing mixed Sc and Sp labeled RNA was done by proportionally scaling the array to a median labeled Sp gene expression level of c. The constant c scales the median half-life of all experiments. We calibrated c in a way that the resulting median Sc wild-type mRNA half-life equaled that observed previously (Miller et al. 2011). Now, all Sc RNA levels, no matter if total or labeled, no matter from which experiment, can be compared on an absolute level. Decay rates and synthesis rates were obtained as described (Miller et al. 2011). The whole analysis workflow has been carried out using the open source R/Bioconductor package DTA

2012-03-28 | E-MTAB-760 | biostudies-arrayexpress

rec-Y2H matrix in S. cerevisiae screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

Project description:RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs recognized by RBPs are typically a poor predictor of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for RBP–RBP complex formation, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our interaction screening discovers RBP pairs that bind RNAs adjacently. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP–RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.

2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress

Transcriptome-wide view of endosomal mRNP transport [RNA-Seq]

Project description:Spatiotemporal protein expression is mediated by active transport and local translation of mRNAs. Key factors of the transport machinery are RNA-binding proteins (RBPs) that recognise mRNAs as cargo for motor-based transport. However, a global view of transported mRNAs with cognate RBP binding sites is missing. Here, we cast a transcriptome-wide view on endosomal mRNP transport in Ustilago maydis by studying the newly identified endosomal RBP Grp1 and the key transport RBP Rrm4 in a comparative iCLIP approach. This uncovered thousand of cargo mRNAs bound by both RBPs.

2018-12-10 | GSE109560 | GEO

Transcriptome-wide view of endosomal mRNP transport [iCLIP ]

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LARP4 Is an RNA-Binding Protein That Binds Nuclear-Encoded Mitochondrial mRNAs To Promote Mitochondrial Function

Project description:Mitochondrial associated RNA-binding proteins (RBPs) have emerged as key contributors to mitochondrial biogenesis and homeostasis. With few examples described, we set out to identify RBPs that post-transcriptionally regulate nuclear-encoded mitochondrial mRNAs (NEMmRNAs). Our analysis of RNA-target sets of RBPs made available by ENCODE identified several RBPs with a preference for binding NEMmRNAs, of which we selected LARP4, a La RBP family member, for further study. We show that LARP4’s RNA-target set is particularly enriched in mRNAs that encode for respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs) across multiple human cell lines. CRISPR knockout cells in conjunction with quantitative proteomics show that protein levels of RCCPs and MRPs are significantly reduced upon loss of LARP4. Furthermore, we show that LARP4 depletion reduces mitochondrial function, and this phenotype is ameliorated by LARP4 re-expression. Our findings shed light onto a novel function for LARP4 as an RBP that binds to NEMmRNAs to promote mitochondrial respiratory function.

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