Multiple 5' terminal cap structures in late polyoma virus RNA.

Nuclear and cytoplasmic polyoma virus-specific RNA extracted from 32P-labeled mouse embryo cells late during productive viral infection was analyzed for the presence of 5' terminal capped structures by complete digestion with RNAases T1, T2 and A, followed by two-dimensional electrophoretic fractionation. Seven major cap I structures (m7 GpppNm1pN2p) were observed in both cases. These termini were further characterized by digestion with penicillium nuclease P1, followed by product analysis in a variety of alternative separate systems. Each structure had an individual combination of N1 and N2 nucleotides, where N1 was always a purine nucleotide but N2 was any nucleotide subject to the single exception that m7GpppGmpCp is found only in low yield. Four different cap II derivatives (m7GpppNm1pNm2pN3p) of four of the cap I structures were also detected in cytoplasmic RNA. None of the termini described derived from contaminating host cell RNA. All of these cap structures mapped on the polyoma viral DNA genome between 66 and 71 map units, a region distant from the 5' end of the bodies of two of the three late polyoma mRNAs. All the polyoma virus-specific cap structures, however, were present in each of the purified 16S, 18S and 19s late mRNAs. These data suggested that families of capped leader sequences of varying sizes are attached to the main body of each late polyoma mRNA species by a splicing mechanism.     

Synthesis of 5' cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase

The 5' end of eukaryotic mRNA carries a N(7)-methylguanosine residue linked by a 5'-5' triphosphate bond. This cap moiety ((7m)GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 5' exonucleases and avoiding its recognition as "nonself" by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 5'-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N(7)-methylguanosine, the synthesis of RNAs possessing a cap structure at the 5' end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 2'-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 5'-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN(2'-Om)-RNAs were purified before the N(7)-methyl group was added by enzymatic means using the human (guanine-N(7))-methyl transferase to yield (7m)GpppN-RNAs (cap-0) or (7m)GpppN(2'-Om)-RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.