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Site-specific 2'-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.
Figure 1. 2’-O-methylation guide activity of Drosophila scaRNA:MeU2-C28 and mutants thereof, detected in injected Xenopus oocytes. (A) Postulated base-pairing of scaRNA:MeU2-C28 with U2 snRNA. (B) Schematic representation of tested MeU2-C28 variants. Experimentally verified guide activity or lack thereof are indicated with a plus or minus sign for each variant. Mutants that lacked the CAB box behaved like the corresponding full-length variants, thus confirming that the CAB box is dispensable for modification guide activity.
Figure 2. Expression of Drosophila snoRNA:Me28S-U3344 and scaRNA:MeU2-C28 (wild-type and single-domain mutant variants) in transfected HeLa cells. (A) Diagram of the snoRNA expression construct used for transfection. (B) Northern blot analysis of MeU2-C28 expression; Me28S-U3344 served as an internal control. Note fully processed guide RNA molecules in all samples. The expression level of the functional single-domain MeU2-C28 guide RNA is similar to that of full-length wild-type. A non-functional, single-domain mutant (extreme mutant with additional mutations 9 and 10) accumulated to a much lower level, yet was still normally processed.
Figure 3. Mapping 2â-O-methylation of Drosophila U2 snRNA in in vitro modification assays. When dNTPs are used at low concentration, reverse transcriptase pauses at 2â-O-methylated positions and short fragments accumulate. The fluorescently labeled fragments appear as peaks above the baseline. The relative height of the peaks is significant only when oocytes from the same frog were used and modification assays were run at the same time (grouped traces). (A) 2â-O-methylation of endogenous Drosophila U2 snRNA (green trace) and in vitro-transcribed Drosophila U2 injected into Xenopus oocytes, either alone (black trace) or with in vitro-transcribed Drosophila MeU2-C28 guide RNA (red trace). Endogenous Drosophila U2 snRNA is modified at positions 48, 41, 28 and 25. When injected into Xenopus oocytes, Drosophila U2 snRNA becomes modified by the endogenous Xenopus modification machinery at position 30, specific to vertebrate U2 snRNA, but not at Drosophila-specific position 28. Vertebrate-specific 2â-O-methylation at positions 11 and 12 is also detected in Drosophila U2 snRNA injected into Xenopus oocytes (black trace). For more details on U2 snRNA modification patterns in vertebrates and Drosophila, see Supplementary Figure S1. Co-injection of MeU2-C28 induces 2â-O-methylation at position 28 (red trace, star). (Aâ) Zoom-in on the region of MeU2-C28-inducible modification. Traces show modification activity of representative mutants of MeU2-C28 guide RNA. Induced 2â-O-methylation at C28 is indicated with a star; unmodified C28 is indicated with an open arrowhead. (B) MeU2-C28 modification activity in RNA-depleted nuclear extract. Wild-type MeU2-C28 (top red trace) is functional in this assay, but the ÎCâ/Dâ mutant (blue trace) and other shorter mutants are not. As previously shown, the ÎCAB mutant is also functional in this assay [13].
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