ABSTRACT
Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.
Subject(s)
Drosophila melanogaster/genetics , Eukaryotic Initiation Factor-4A/genetics , Polytene Chromosomes/chemistry , RNA Precursors/genetics , RNA Splicing , RNA, Messenger/genetics , Animals , Cell Fractionation , Cells, Cultured , Chromosome Mapping , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Exons , Introns , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polytene Chromosomes/metabolism , Protein Binding , RNA Precursors/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Premature translation termination leads to a reduced mRNA level in all types of organisms. In eukaryotes, the phenomenon is known as nonsense-mediated mRNA decay (NMD). This is commonly regarded as the output of a specific surveillance and destruction mechanism that is activated by the presence of a premature translation termination codon (PTC) in an atypical sequence context. Despite two decades of research, it is still unclear how NMD discriminates between PTCs and normal stop codons. We suggest that cells do not possess any such mechanism and instead propose a new model in which this mRNA depletion is a consequence of the appearance of long tracts of mRNA that are unprotected by scanning ribosomes.
Subject(s)
Codon, Nonsense/genetics , Nonsense Mediated mRNA Decay/genetics , Peptide Chain Termination, Translational , RNA, Messenger/genetics , Codon, Terminator/genetics , Ribosomes/geneticsABSTRACT
The nucleolus is the most prominent morphological feature within the nucleus of eukaryotic cells and is best known for its role in ribosome biogenesis. It forms around highly transcribed ribosomal RNA gene repeats which yield precursor rRNAs that are co-transcriptionally processed, folded and, while still within the nucleolus, associate with most of the ribosomal proteins. The nucleolus is therefore often thought of as a factory for making ribosomal subunits, which are exported as inactive precursors to the cytoplasm where late maturation makes them capable of mRNA binding and translation initiation. However, recent studies have shown substantial evidence for the presence of functional, translation competent ribosomal subunits within the nucleus, particularly in the nucleolus. These observations raise the intriguing possibility that the nucleolus, as well as being a ribosome factory, is also an important nuclear protein-synthesis plant.
