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In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2'-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.
FIGURE 1. Testing SNORA24 modification guide activities. (A) Mapping of pseudouridines in 18S rRNA from human HeLa cells (red trace) and Xenopus laevis XTC cells (green and blue traces) using fluorescent primer extension. Peaks corresponding to reverse transcription termination at modified positions are indicated. Arrowheads point to unmodified positions. Ectopic expression of human SNORA24 in XTC cells induces pseudouridylation of Xenopus 18S rRNA at position 574, equivalent to human 18S-609 (blue trace, star). Postulated base-pairing between human SNORA24 and 18S rRNA shown in the top right corner. (B) Predicted base-pairing between Xenopus SNORA24 and 18S rRNA (left) and Pus7p recognition motif (right). (C) Modification of the artificial substrate RNA specific for xtSNORA24 (U87-xt18S[1600–1620]) in wild type BY4741and pus7Δ mutant yeast strains. The position corresponding to xt18S-1607 in the artificial substrate RNA is pseudouridylated in wild type (black trace) and in the pus7Δ strain that expresses xtSNORA24 (green trace, star). This position is not modified in the pus7Δ strain itself (blue trace, arrowhead) and in the pus7Δ strain that expresses Xenopus Pus7p (pink trace, arrowhead). Note, that in a fragment of 18S rRNA [1600–1620], yeast Pus7p recognizes a sequence different from the consensus UGΨAR (Urban et al. 2009; Carlile et al. 2014; Schwartz et al. 2014): It contains A instead of unvarying U at −2 position, AGΨAA (B). (D) Pseudouridylation of yeast U2 snRNA in wild type (black trace) and mutant pus7Δ strains (blue and pink traces). Expression of Xenopus Pus7p in the pus7Δ yeast strain rescues pseudouridylation of U2 snRNA at position 35 (pink trace, star).
FIGURE 2. (A) Predicted base-pairing of SNORA18, SNORA22, and SNORA71 with 18S rRNAs at position 1720 and with 28S rRNA at positions 4501 and 4502, respectively. (B,C) Pseudouridylation of artificial substrate RNAs in yeast cells. (B) Yeast optimized SNORA18 (C-to-U-mutant) can induce predicted pseudouridylation in a fragment of yeast 18S rRNA [1647–1668 nt] inserted in U87 RNA (pink trace, star). Expression of the artificial substrate RNA U87-y18S[1647–1668] alone used as a negative control (blue trace). (C) When expressed in yeast cells, human SNORA22 (red trace, red star) and Xenopus SNORA22(2) (dark green trace) can induce pseudouridylation of position 4501 in a 28S rRNA fragment [4491–4508 nt] inserted in U87 RNA. Xenopus SNORA22(1) (light green trace, arrowhead) and SNORA57 (dark blue trace, arrowhead) do not show modification activity on the same substrate RNA. SNORA57 activity on 28S-4501 was predicted by Kehr et al. (2014); for the proposed base-pairing see Supplemental Figure S1. Note that two copies of Xenopus SNORA22—functional and nonfunctional—differ only in their upper stem structures. In human, 28S rRNA position 4502 is pseudouridylated, and this position becomes modified in the artificial substrate RNA expressed in HeLa cells (top gray trace, black star). SNORA71 can base-pair with 28S rRNA at position 4502 (A). However, when it was tested in yeast, SNORA71 could not induce this modification in the artificial substrate RNA (light blue trace, arrowhead).
FIGURE 3. 18S rRNA modification under normal and high temperature conditions in yeast (A,C) and amphibian cells (D,E). (A) Yeast optimized SNORA18 (C-to-U mutant, depicted in Fig. 2A) does not induce pseudouridylation of yeast 18S rRNA at position 1656 (equivalent to human 18S-1720) in the BY4741 strain grown at 30°C, nor can it do so at 37°C (blue traces). “Perfect” SNORA18, a “perfect guide” RNA for position 1656 in yeast 18S rRNA made from SNORA18 (depicted in Fig. 2A), induces pseudouridylation of the target position both at 30°C (not shown) and 37°C (top pink trace, star). Note there is no Pus7p activity on position 1585 in 18S rRNA both at 30°C and 37°C. Positions that were expected to be modified are indicated with arrowheads. (B) Postulated base-pairing of X. tropicalis and X. laevis SNORA28 with 18S rRNA. For the 5′-terminal (left schematic) and the 3′-terminal pseudouridylation pockets (right schematic), target positions and corresponding modifications are indicated on the right side of each pocket. A mismatched nucleotide in the 3′-terminal X. laevis ASE is highlighted in green. (C) Yeast cells grown at 37°C do not induce pseudouridylation activity of X. laevis SNORA28 on yeast 18S rRNA at position 808 (equivalent to Xenopus 18S-828). Note the increased modification level at position 759, the other target of SNORA28 (light blue trace), as compared to controls (gray and red traces). X. tropicalis SNORA28 is functional on position 808 (top dark blue trace, star). (D,E) X. laevis XTC cells (D) and axolotl embryos (E) grown at elevated temperatures do not have altered pseudouridylation patterns of 18S rRNA. (D) When snoRNA with a functional pseudouridylation pocket for 18S-828 (xtSNORA28 > U2-Ψ60, blue trace) is expressed in XTC cells, this position is modified (star). Unmodified position 828 is indicated with arrowheads.
FIGURE 4. SNORA57 guide activities on yeast 18S rRNA. (A) Postulated base-pairing of SNORA57 with human (red) and yeast (blue) 18S rRNA; the 5′ terminal pseudouridylation pocket is shown on the left and the 3′-terminal pseudouridylation pocket on the right. To make a yeast optimized version of SNORA57 an indicated U-to-C mutation was introduced in the ASE of the 5′ terminal pseudouridylation pocket. (B) Mapping pseudouridines in human 18S [968–1086 nt] (HeLa, top gray trace) and the equivalent region of yeast 18S rRNA (BY4741, black, red, and blue traces). Normally, yeast 18S rRNA has only Ψ999 in this region (black trace). Expression of vertebrate SNORA57 did not change the modification pattern of yeast 18S rRNA (red trace, arrowheads indicate predicted target positions for SNORA57). Yeast optimized SNORA57 with the U-to-C mutation could induce pseudouridylation of yeast 18S rRNA at two positions, 947 and 989 (blue trace, stars).
FIGURE 5. (A) A pseudouridylation pocket generated in xtSCARNA4 and SNORA28 to make artificial guide RNAs for Xenopus U2 snRNA modification at position 60 (SCARNA4 > U2-Ψ60 and xtSNORA28 > U2-Ψ60). Mutations that shorten the ASE are indicated in blue; a fragment of U2 snRNA inserted in a U87-based artificial substrate RNA is underlined. (B) Mapping pseudouridines in human and Xenopus U2 snRNA. Normally, human U2 snRNA (HeLa, gray trace) is pseudouridylated at position 60; Xenopus U2 snRNA is not modified at this position (XTC, green trace). Expression of SCARNA4 > U2-Ψ60 (red trace, star) but not xtSNORA28>U2-Ψ60 (dark blue trace, arrowhead) induced U2 snRNA pseudouridylation at position 60 in the Xenopus laevis cell line XTC. (C) Pseudouridylation of the artificial substrate RNA containing a fragment of vertebrate U2 snRNA (U87-U2[51–68]) in yeast cells. Even with a short ASE, xtSNORA28 > U2-Ψ60 is functional on U2 snRNA artificial substrate (blue trace, star).
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