Abstract
Until cytoplasmic recapping was discovered, decapping was thought to irreversibly destine an mRNA to degradation. Contradicting this idea, we readily observe mRNAs targeted by cytoplasmic capping in uncapped, yet stable forms. 5’ RACE shows that nearly all uncapped ends correspond to CAGE tags and that the recapping of ZNF207 mRNA may be restricted to a single splice isoform. A modified RACE approach detected uncapped 5’ RNA ends mapping to 46 mRNAs in dominant negative cytoplasmic capping enzyme expressing and normal cells. 11 of 46 cloned mRNAs also contained splice isoform-limiting sequences. Collectively, these data reinforce earlier work and suggest that alternative splicing may play a role in targeting transcripts for– and/or determining the position of– cytoplasmic capping.
- List of Abbreviations
- m7G
- N7-methylguanosine cap
- RNMT
- RNA guanine-7 methyltransferase
- NMD
- nonsense-mediated decay
- CAGE
- capped analysis of gene expression
- RACE
- rapid amplification of cDNA ends
- IPTG
- Isopropyl β-D-1- thiogalactopyranoside
- X-Gal
- 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
- K294A
- dominant negative cytoplasmic capping enzyme