Abstract
In metazoa cap 1 (m(7)GpppNmp-RNA) is linked to higher levels of translation; however, the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m(7)GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m(7)Gpppm(6,6)AmpAmpCmpm(3)Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives an m(2,2,7)G cap with hypermethylation at position one only (m(2,2,7)Gpppm(6,6)AmpApCpUp-U1 snRNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg(2+). Positions Lys(95)-Asp(204)-Lys(259)-Glu(285) constitute the conserved catalytic core. A guanosine cap on RNA independent of its N(7) methylation status is required for substrate recognition, but an m(2,2,7G)-cap is not recognized. TbMTr1 favors the spliced leader 5' sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification.
Highlights
Most mature eukaryotic and viral mRNAs possess a 5Ј cap that consists of an inverted guanosine methylated at position N7 linked to the first transcribed RNA nucleotide by a unique 5Ј-5Ј triphosphate bond (m7GpppN)
Purification and Activity of rTbMTr1—TbMTr1-null T. brucei show an absence of cap 1 modification on spliced leader (SL) RNA and U1 snRNA [18]. rTbMTr1 was produced in bacteria to facilitate detailed studies on the enzyme biochemistry, structure, and function
Based on our bioinformatics analyses and because Autographa californica nucleopolyhedrosis virus (AcNPV) orf 69 encodes a functionally characterized cap 1 2Ј-O-ribose MTase [22], we propose a similar structure for TbMTr1 and its homologs
Summary
Most mature eukaryotic and viral mRNAs possess a 5Ј cap that consists of an inverted guanosine methylated at position N7 linked to the first transcribed RNA nucleotide by a unique 5Ј-5Ј triphosphate bond (m7GpppN). To test MTase activity of the purified protein a 150-nucleotide synthetic RNA with a methylated G-cap (m7G*pppApGpSL) was used as substrate.
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