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.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.