Post-transcriptional modification of spliceosomal RNAs is normal in SMN-deficient cells

Abstract

The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 2'-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 2'-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.

FIGURE 1.

Mapping of 2′-O-methylated nucleotides (A) and pseudouridines (B,C) in human U2 snRNA using radioactive primer extension. Filled arrowheads indicate stop signals corresponding to known modified residues; open arrowheads in B and C indicate the newly recognized pseudouridine at position 60. (A) Primer extension was performed in the presence of 1 mM and 0.004 mM dNTP on RNA isolated from control HeLa cells (SMN) and from HeLa cells depleted for SMN using siRNA (SMNΔ). (Lanes GAUC) Dideoxy sequencing reactions. (B) Total RNA from control (SMN) and SMN-depleted (SMNΔ) HeLa cells was either treated with CMC and alkali buffer (lanes Ψ) or with alkali buffer alone as a control (lanes N). (Lanes GAUC) Sequencing reactions. (C) Primer extensions were performed as described in B using RNA isolated from SMN1^−/− fibroblasts (−/−) and heterozygous controls (+/−) as well as from SMN1^−/− lymphoblasts.

FIGURE 2.

Mapping of modified nucleotides in U2 snRNA from SMN1^−/− and control SMN1^+/+ lymphoblasts using the fluorescent primer extension technique. Positions of nucleotides were determined by alignment with sequencing reactions run on in vitro-transcribed U2 snRNA (shown at top). (A) Twelve pseudouridines are detectable in human U2 snRNA from SMN1^+/+ (red traces) and SMN1^−/− lymphoblasts (blue traces) after CMC treatment (Ψ6, Ψ7, Ψ15, Ψ34, Ψ37, Ψ39, Ψ41, Ψ43, Ψ44, Ψ54, Ψ58, Ψ60). (*) The newly identified Ψ60. Peaks corresponding to Ψ6, Ψ7, and Ψ15 are more prominent when samples are overloaded (inset). CMC-treated in vitro-transcribed U2 shows no internal peaks (black). (B) Seven 2′-O-methylated residues are detectable in human U2 from SMN1^+/+ (red traces) and SMN1^−/− lymphoblasts (blue traces) when primer extension is performed at a low concentration of dNTPs using AMV-RT (Am1, Gm11, Gm12, Gm25, Am30, Cm40, Um47). No peaks are observed when primer extension is performed on in vitro-transcribed U2 snRNA (black). (C) Nine 2′-O-methylated residues are detectable unequivocally when SuperScript 2 RT was used for primer extension (Am1, Um2, Gm11, Gm12, Gm19, Gm25, Am30, Um47, Cm61). Note the double stop signal for Cm61: one typical stop preceding the 2′-O-methylated nucleotide and the second stop at the actual position of the modification. In total, all known 2′-O-methylated positions were detected in B and C. (D) Control reactions were performed at a high concentration of dNTPs using RNA from SMN1^+/+ (red) and SMN1^−/− (blue) lymphoblasts without any chemical treatment.

FIGURE 3.

Mapping of modified nucleotides in minor snRNAs U12 (left) and U4atac (right). (A) Three 2′-O-methyl groups are detectable in U12 snRNA from SMN1^+/+ (red) and SMN1^−/− (blue) lymphoblasts (Am8, Gm18, Gm22). (B) Two pseudouridines are found in U12 snRNA from SMN1^+/+ (red) and SMN1^−/− (blue) lymphoblasts (Ψ19 and Ψ28) and no peak is observed for in vitro-transcribed U12 snRNA (black). (C) Only a tiny peak for Am8 is detectable in control untreated RNA. (D) 2′-O-methylation is detectable in U4atac at positions A1, A2, an G19. A prominent stop is observed also at position 9 (star) suggesting a 2′-O-methyl group at C8, but the reaction is terminated at the same position when in vitro-transcribed RNA is used (black trace). (E) A pseudouridine at position 12 is detectable in U4atac from SMN1^+/+ (red) and SMN1^−/− (blue) lymphoblasts, but not in control in vitro-transcribed U4atac (black). (F) When control primer extension reactions are run on RNA samples isolated from lymphoblasts an extra peak appears at position 5. This might correspond to a different isoform of human U4atac. In fact, 19 different genes are reported for U4atac in the human genome databases (http://useast.ensembl.org/Homo_sapiens/) and the U4atac.14-201 variant fits the detected size.