Interference probing of rRNA with snoRNPs: A novel approach for functional mapping of RNA in vivo

Abstract

Synthesis of eukaryotic ribosomal RNAs (rRNAs) includes methylation of scores of nucleotides at the 2-O-ribose position (Nm) by small nucleolar RNP complexes (snoRNPs). Sequence specificity is provided by the snoRNA component through base-pairing of a guide sequence with rRNA. Here, we report that methylation snoRNPs can be targeted to many new sites in yeast rRNA, by providing the snoRNA with a novel guide sequence, and that in some cases growth and translation activity are strongly impaired. Novel snoRNAs can be expressed individually or by a unique library strategy that yields guide sequences specific for a large target region. Interference effects were observed for sites in both the small and large subunits, including the reaction center region. Targeting guide RNAs to nucleotides flanking the sensitive sites caused little or no defect, indicating that methylation is responsible for the interference rather than a simple antisense effect or mis-guided chaperone function. To our knowledge, this is the only approach that has been used to mutagenize the backbone of rRNA in vivo.

Mis-targeted methylation in rRNA can severely impair ribosome synthesis and activity

“…Consistent with this hypothesis, our previous data showed that the deleterious snoRNAs usually under-accumulate. 5,6 Taken together, the results demonstrate that mis-targeting methylation can impair both ribosome synthesis and function. The growth defects are most likely due to methylation rather than an antisense interference effect caused by the new snoRNP, as evidenced by these Figure 4.…”

“…We have also observed similarly sharp positional effects for two other sensitive sites examined in both 18S and 25S rRNAs. 6,7 Our discovery that novel methylations can trigger severe rRNA degradation shows that modification or snoRNP effects are monitored during ribosome synthesis by some surveillance system(s), either directly or indirectly. Consistent with this view, it has been shown in Schizosaccharomyces pombe that substitution mutations at several positions in the PTC region that are normally methylated or pseudouridylated also caused degradation of rRNA.…”

“…Interference was proposed to be caused by the methylation mutation, rather than an antisense effect, since targeting nearby nucleotides in this and other sensitive rRNA regions had little or no effect. 6 In the present study, we characterize the basis of growth defects observed from methylation probing of eight sites in the PTC region predicted to be functionally important 6,8,9,11,12 (Fig. 1A).…”

“…Using this approach, we previously probed the peptidyl transferase center (PTC) region of the large subunit rRNA, which includes the reaction center. In most cases, targeting a novel site had no apparent growth effect, however, severe growth defects were observed for five positions, 6,7 all at or adjacent to sites involved in tRNA binding or peptidyl transferase (PTase) activity. [8][9][10] These findings suggest that the mis-targeted methylations interfere with PTC function.…”

Regulation of pre-mRNA splicing inXenopusoocytes by targeted 2′-O-methylation

“…Interestingly, when expressed in the cell, artificial snoRNAs can also efficiently guide rRNA modifications at target sites (Cavaille et al 1996;Ni et al 1997;Liu et al 2001Liu et al , 2008Liu and Fournier 2004).…”