Abstract
Author SummaryWe previously described a cyclical process of mRNA decapping and recapping termed “cap homeostasis.” Recapping is catalyzed by a complex of cytoplasmic proteins that includes the enzyme known to catalyze nuclear capping, and a kinase that converts RNA with a 5′-monophosphate end to a 5′-diphosphate capping substrate. The current study shows these two enzymatic activities are brought together in the cytoplasmic capping complex as both bind to adjacent domains of the adapter protein Nck1. Nck1 is a cytoplasmic protein best known for transducing receptor tyrosine kinase signaling. We identify a proline-rich sequence at the C-terminus of a human capping enzyme that is required for binding to Nck1, and we show that this interaction is required for integrity of the cytoplasmic capping complex. Depletion of Nck1 causes the cytoplasmic capping complex to dissociate. The inhibition of cytoplasmic capping by Nck1 with mutations in either the 5′-kinase or capping enzyme binding sites identified a functional role for Nck1 in cap homeostasis and a previously unknown function for Nck1 in cell biology.
Highlights
The 7-methylguanosine ‘‘cap’’ is a defining feature of all eukaryotic mRNAs, and the cap plays a role in almost every step of mRNA metabolism
We previously described a cyclical process of mRNA decapping and recapping termed ‘‘cap homeostasis.’’ Recapping is catalyzed by a complex of cytoplasmic proteins that includes the enzyme known to catalyze nuclear capping, and a kinase that converts RNA with a 59monophosphate end to a 59-diphosphate capping substrate
The original purpose of that study was to identify mRNAs that are regulated by cytoplasmic capping, and in the course of doing so we discovered a cyclical process of decapping and recapping that we termed ‘‘cap homeostasis.’’ Cytoplasmic capping targets can be grouped into three categories on the basis of their cap status and stability in cells that are inhibited for cytoplasmic capping
Summary
The 7-methylguanosine ‘‘cap’’ is a defining feature of all eukaryotic mRNAs, and the cap plays a role in almost every step of mRNA metabolism. The cap is bound by a heterodimer of CBP80-CBP20, and its interaction with other proteins coordinates many of the subsequent steps in pre-mRNA processing and mRNA surveillance [1]. Translation and mRNA decay are interconnected processes, and for many transcripts loss of the cap is thought to be an irreversible step leading to mRNA decay [2]. A number of approaches use the cap to map transcription start sites. A significant number of CAGE tags do not correspond to transcription start sites [8], mapping instead to locations within the body of the transcript. There is no evidence for downstream CAGE tags in the Drosophila transcriptome [6], suggesting that the presence of capped ends located downstream within the transcript body is unique to higher metazoans
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