Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing - PubMed (original) (raw)
Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing
Xue-Hai Liang et al. RNA. 2009 Sep.
Abstract
The ribosome decoding center is rich in modified rRNA nucleotides and little is known about their effects. Here, we examine the consequences of systematically deleting eight pseudouridine and 2'-O-methylation modifications in the yeast decoding center. Loss of most modifications individually has no apparent effect on cell growth. However, deletions of 2-3 modifications in the A- and P-site regions can cause (1) reduced growth rates (approximately 15%-50% slower); (2) reduced amino acid incorporation rates (14%-24% slower); and (3) a significant deficiency in free small subunits. Negative and positive interference effects were observed, as well as strong positional influences. Notably, blocking formation of a hypermodified pseudouridine in the P region delays the onset of the final cleavage event in 18S rRNA formation ( approximately 60% slower), suggesting that modification at this site could have an important role in modulating ribosome synthesis.
Figures
FIGURE 1.
The decoding region of yeast rRNA is rich in modifications. (A) Secondary structure of the decoding center of 18S rRNA. Modified nucleotides in the decoding center are highlighted by broken boxes, and the corresponding guide snoRNAs are also identified. Methylations (●); pseudouridines (△). (B) The 3D structure of modifications in the decoding center. Locations are deduced from the ribosome structure. The corresponding nucleotide numbers in E. coli rRNA are provided in parentheses. The system of representing modifications with colors is the same in both panels. The modifications in the A, Aa, P, and E regions are indicated by pink, yellow, green, and red circles, respectively. The lower left panel shows a schematic cartoon for positions of A-region (white), Aa-region (gray), and P-region (black) modifications. This system is used in subsequent figures to indicate modification situations.
FIGURE 2.
Depletion of decoding region modifications can impair cell growth and increase cell sensitivity to neomycin. (A) Growth comparisons for strains depleted of P-region Ψ (−1a), Aa-region Ψ (−1b), and Aa-region Nm (−1c) individually or in different combinations. Two OD600 units of cells were diluted serially (1:5), spotted on plates with rich YPD medium in the presence (right panel) or absence (left panel) of neomycin (0.4 mg/mL), and incubated at different temperatures. (B) Growth properties of strains depleted of Aa-region Nm (−1c) and P-region Nm (−1d) individually or in different combinations, as in A. Strain (−)1e is depleted of an A-region Nm (Gm1271). Strains showing cold-sensitive phenotypes (boxed) and increased neomycin sensitivity (arrowheads) are identified. The schematic structures at the left of the data panels indicate the modification situations, as shown in Figure 1B.
FIGURE 3.
Abnormal polysome profiles can occur with loss of modifications. Extracts from log phase cells were fractionated on sucrose gradients and polysome patterns detected by UV absorbance at 254 nm. Peaks are identified for control cells. Increases in the relative abundance of 60S complexes are marked (*).
FIGURE 4.
Altered growth and polysome properties can be fully restored by complementation with the missing snoRNAs. (A) Growth was compared at 16°C for control and test cells containing an empty vector and test cells expressing plasmid-encoded snoRNAs (+), as indicated. The culture conditions were as in Figure 2, except that the medium lacked adenine. (B) Polysome profiles were examined as described in Figure 3.
FIGURE 5.
Depletion of snR35 blocks the formation of the hypermodified pseudouridine. (A) Biosynthetic pathway of the m1acp3Ψ hypermodification (Brand et al. 1978). (B) Primer extension analysis does not reveal the pseudouridylation status of the hypermodified site. RNA treated or not with CMC was subjected to primer extension analysis, with reaction products separated on an 8% polyacrylamide gel. (C) The chromatographic behavior is altered in cells depleted of snR35. An excised [32P]18S rRNA fragment containing the hypermodified site and two additional pseudouridines from control (Con.) or (−)1a cells depleted of snR35 was digested by nuclease P1 and analyzed with TLC. The identities of the different nucleotide spots are indicated.
FIGURE 6.
Disrupting the hypermodification strongly delays hydrolytic processing of pre-rRNA. (A) Schematic representation of the yeast pre-rRNA processing pathway. Cleavage sites involved in formation of 18S rRNA are shown. The processing steps for 27S pre-rRNA are depicted in a simplified form. (B) Radiolabeling pulse-chase analysis shows that pre-rRNA processing is delayed in cells lacking the hypermodified Ψ. Pre-rRNA was labeled in vivo using [3H]methionine, and processing products were analyzed on 1.2% agarose gels and visualized with a PhosphorImager. The chase times (minutes) are indicated above the lanes. (C) Percentages of converted mature 18S rRNA (upper panel) and 25S rRNA (lower panel) at each time point. Signal strength was measured from the results in panel B using ImageJ, and the calculated values plotted. (D) Loss of the hypermodified Ψ causes substantial accumulation of 20S pre-rRNA. Steady state levels of rRNA species were examined by Northern hybridization, with oligonucleotides specific to 20S and 27S pre-rRNAs. U2 snRNA was used as a control for sample loading. (E) The 20S pre-rRNA lacking the hypermodified Ψ is exported to the cytoplasm. The presence of the cytoplasmic dimethylations in the 20S pre-rRNA was detected by primer extension analysis. The reaction products were separated on an 8% polyacrylamide gel, next to a sequencing ladder created with the same oligonucleotide.
FIGURE 7.
The snoRNA (snR35) requirement can be satisfied with a different snoRNA containing the corresponding Ψ guide elements. (A) Structure of the hybrid snoRNA created by replacing the Ψ guide elements in snR36 snoRNA with those from snR35. The substituted nucleotides in the hybrid snoRNA are shown in bold and the predicted interactions between the mutant snoRNA and pre-rRNA are given. (B) The hybrid RNA can fully complement loss of the snR35 snoRNA. Growth properties were compared on solid medium for the (−)3b test strain transfromed with expression plasmids for hybrid and parental snoRNAs. (C) The impaired polysome pattern was restored to normal by the mutant snoRNA. Polysome patterns are shown for two mutant strains (−1a and −3b) expressing or not the hybrid snoRNA. (D) A two-nucleotide deletion in snR35 inactivates its modification function. The nucleotides deleted are shown in bold. (E) The modification-defective mutant snoRNA (snR35m) does not restore cell growth. Growth properties were compared for (−)3b test cells expressing a plasmid-borne deletion mutant snR35 (snR35m), or control guide snoRNAs (snR56 and snR70). (F) The impaired polysome pattern in (−)3b cells could not be restored to normal by expressing the inactive snR35 (snR35m) or natural snR36. Control, Ys602 cells with an empty vector.
References
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- Brand RC, Klootwijk J, Van Steenbergen TJ, De Kok AJ, Planta RJ. Secondary methylation of yeast ribosomal precursor RNA. Eur J Biochem. 1977;75:311–318. - PubMed
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