ABSTRACT
Little is known about crosstalk between the eukaryotic transcription and translation machineries that operate in different cell compartments. The yeast proteins Rpb4p and Rpb7p represent one such link as they form a heterodimer that shuttles between the nucleus, where it functions in transcription, and the cytoplasm, where it functions in the major mRNA decay pathways. Here we show that the Rpb4/7 heterodimer interacts physically and functionally with components of the translation initiation factor 3 (eIF3), and is required for efficient translation initiation. Efficient translation in the cytoplasm depends on association of Rpb4/7 with RNA polymerase II (Pol II) in the nucleus, leading to a model in which Pol II remotely controls translation. Hence, like in prokaryotes, the eukaryotic translation is coupled to transcription. We propose that Rpb4/7, through its interactions at each step in the mRNA lifecycle, represents a class of factors, "mRNA coordinators," which integrate the various stages of gene expression into a system.
Subject(s)
Gene Expression Regulation, Fungal , Protein Biosynthesis , RNA Polymerase II/metabolism , RNA Stability , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription, Genetic , DNA-Directed RNA Polymerases/metabolism , Eukaryotic Initiation Factor-3/metabolism , Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
During eukaryotic translation, mRNAs may form intra-molecular interactions between distant domains. The 5'-cap and the polyA tail were shown to interact through their associated proteins, and this can induce physical compaction of the mRNA in vitro. However, the stability of this intra-molecular association in translating mRNAs and whether additional contacts exist in vivo are largely unknown. To explore this, we applied a novel approach in which several endogenous polysomal mRNAs from Saccharomyces cerevisiae were cleaved near their stop codon and the resulting 3'-UTR fragments were tested either for co-sedimentation or co-immunoprecipitation (co-IP) with their ORFs. In all cases a significant fraction of the 3'-UTR fragments sedimented similarly to their ORF-containing fragments, yet the extent of co-sedimentation differed between mRNAs. Similar observations were obtained by a co-IP assay. Interestingly, various treatments that are expected to interfere with the cap to polyA interactions had no effect on the co-sedimentation pattern. Moreover, the 3'-UTR appeared to co-sediment with different regions from within the ORF. Taken together, these results indicate extensive physical associations between 3'-UTRs and their ORFs that vary between genes. This implies that polyribosomal mRNAs are in a compact configuration in vivo.
Subject(s)
3' Untranslated Regions/chemistry , Open Reading Frames , 3' Untranslated Regions/isolation & purification , 3' Untranslated Regions/metabolism , Centrifugation, Density Gradient , Immunoprecipitation , Poly A/metabolism , Polyribosomes/chemistry , Potassium Chloride/chemistry , Protein Biosynthesis , RNA Caps/metabolism , RNA, Messenger/isolation & purification , Saccharomyces cerevisiae/geneticsABSTRACT
The number and distribution of ribosomes on a transcript provide useful information in ascertaining the efficiency of translation. Herein we describe a direct method to determine the association of ribosomes with specific regions of an mRNA. The method, termed Ribosome Density Mapping (RDM), includes cleavage of ribosomes-associated mRNAs with RNase H and complementary oligodeoxynucleotide followed by separation of the cleavage products on a sucrose gradient. The gradient is then fractionated and the sedimentation position of each mRNA fragment is determined by northern analysis. Although developed for yeast mRNAs, RDM is likely to be applicable to various other systems.
Subject(s)
Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Ribosomes/metabolism , Blotting, Northern , Centrifugation, Density Gradient , Molecular Biology/methods , RNA, Fungal/genetics , RNA, Fungal/isolation & purification , Ribonuclease HABSTRACT
In eukaryotes, scanning of the 5' leader by the small ribosomal subunit precedes recognition of the start codon. Thus, various sequence elements that are located within this region may affect ribosomes' progression and lead to significant effects on translation. Most notable are short ORFs located upstream of the start codon, which are known to regulate the translation of the main ORF in the transcript. The function of these elements is likely to correlate with altered ribosomal association with the 5' leader of the mRNA. Currently, the only method to determine the ribosomal association of different regions of the mRNA in vivo is the Ribosome Density Mapping (RDM) procedure. This method entails cleavage of the target mRNA by specific oligodeoxynucleotides and RNase H and separation of the cleavage products by velocity sedimentation in a sucrose gradient. In this chapter, we provide a detailed protocol for this procedure and discuss its feasibility.
Subject(s)
5' Untranslated Regions/analysis , 5' Untranslated Regions/metabolism , Centrifugation, Density Gradient/methods , Ribosomes/metabolism , Animals , Humans , Models, Biological , Polyribosomes/chemistry , Ribonuclease H/metabolismABSTRACT
Missense mutations of the ligand binding domain of hepatocyte nuclear factor (HNF)-4alpha result in maturity onset diabetes of the young (MODY)-1. We show here that MODY-1 as well as Gln-185 missense mutants of the ligand binding domain of HNF-4alpha fail to transactivate transcription of HNF-4alpha-responsive genes. Defective transactivation by these mutants is accounted for by their reduced binding affinities for fatty acyl agonist ligands of HNF-4alpha. These mutants may be rescued by exogenous fatty acid agonist ligands of HNF-4alpha, yielding transcriptional activities in the wild type range. The effect of added ligands is synergistic with that of transcriptional coactivators of HNF-4alpha. These findings may indicate the means for treating selected MODY-1 subjects with HNF-4alpha agonist nutrients and drugs.