Abstract
Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.
Highlights
Termed interchromatin granule clusters (IGCs) due to the observation of 20–25 nm nuclear granules in electron microscopy micrographs [1], nuclear speckles are dynamic nuclear bodies (∼20–30 per nucleus) that are enriched in RNA binding proteins (RBPs) [2,3]
We found that many nuclear proteins and RNAs were mislocalized under these stress conditions, components of the nuclear pore complex (NPC) and RNA processing factors that are necessary for mRNA export
Since tubercidin is a toxic adenosine analogue that interferes with translation, we considered the possibility that it may be lead to stress granule (SG) formation, cytoplasmic structures that are induced upon several cellular stresses and contain untranslated mRNAs and various RBPs [30,31]
Summary
Termed interchromatin granule clusters (IGCs) due to the observation of 20–25 nm nuclear granules in electron microscopy micrographs [1], nuclear speckles are dynamic nuclear bodies (∼20–30 per nucleus) that are enriched in RNA binding proteins (RBPs) [2,3]. Prominent examples include the SR proteins with roles in pre-mRNA splicing, mRNA export, RNA stability and translation [4,5,6,7]. Because RBPs are not retained in speckles, they are able to diffuse to active sites of transcription, where they participate in splicing. They may be recruited from nuclear speckles to pre-mRNA in the nucleoplasm during post-transcriptional splicing, mRNP formation and/or mRNA export [2,8,9]
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