Daniel R. Schoenberg, Center for RNA Biology and Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH

Cytoplasmic capping: A new post-transcriptional step that may expand the transcriptome and the proteome

The addition of a cap to the 5’-end is the first post-transcriptional modification made to newly synthesized pre-mRNAs, and the cap plays a key role in nuclear processing, export, surveillance, translation, storage and decay. It was generally thought that addition of the cap is restricted to the nucleus and loss of the cap is irreversible, leading directly to mRNA decay. Results from our lab challenge this notion, and indicate that cytoplasmic capping may add a new dimension to the transcriptome. In the course of studying the fate of nonsense-containing b-globin mRNA we reexamined results from 1992 that the products of its decay were modified with a 5’ cap or cap-like structure. This led to the identification of a previously unrecognized population of cytoplasmic capping enzyme as the likely effector.  In nuclear capping the 5’-triphosphate end on newly-synthesized pre-mRNA is hydrolyzed to a diphosphate, and GMP bound covalently to capping enzyme is transferred onto this to generate the GpppX cap. This is then methylated to complete the cap structure. Caps are removed from cytoplasmic RNA by either decapping or by endonuclease cleavage, both of which produce RNAs with a 5’-monophosphate end.  In order to be re-capped this must be converted to a 5’-diphosphate, and no such enzyme had been reported. I will describe the identification and properties of a cytoplasmic complex that contains capping enzyme and this kinase, discuss the relationship of this to the storage of non-translating mRNAs in discrete foci (P bodies), and show that interfering with cytoplasmic capping reduces the ability of cells to recover from stress. Our findings coincide with results of deep sequencing that identified a new class of capped small RNAs (termed non-PASRs) that are products of fully processed transcripts missing sequences from their 5’ ends. Moreover, the discovery of dynamic populations of uncapped transcripts in Arabidopsis suggests the possibility that re-capping may be a general mechanism for reactivating the translation mRNAs that are stored or silenced by microRNAs. Although at thi point it is highly speculative, cytoplasmic capping has the potential to expand the proteome by enabling the translation of N-terminally-truncated proteins from mRNAs missing sequences from their 5’ ends.

Supported by grants from the NIH