Mapping of ribosomal 23S ribosomal RNA modifications in Clostridium sporogenes

Abstract

All organisms contain RNA modifications in their ribosomal RNA (rRNA), but the importance, positions and exact function of these are still not fully elucidated. Various functions such as stabilizing structures, controlling ribosome assembly and facilitating interactions have been suggested and in some cases substantiated. Bacterial rRNA contains much fewer modifications than eukaryotic rRNA. The rRNA modification patterns in bacteria differ from each other, but too few organisms have been mapped to draw general conclusions. This study maps 23S ribosomal RNA modifications in Clostridium sporogenes that can be characterized as a non-toxin producing Clostridium botulinum. Clostridia are able to sporulate and thereby survive harsh conditions, and are in general considered to be resilient to antibiotics. Selected regions of the 23S rRNA were investigated by mass spectrometry and by primer extension analysis to pinpoint modified sites and the nature of the modifications. Apparently, C. sporogenes 23S rRNA contains few modifications compared to other investigated bacteria. No modifications were identified in domain II and III of 23S rRNA. Three modifications were identified in domain IV, all of which have also been found in other organisms. Two unusual modifications were identified in domain V, methylated dihydrouridine at position U2449 and dihydrouridine at position U2500 (Escherichia coli numbering), in addition to four previously known modified positions. The enzymes responsible for the modifications were searched for in the C. sporogenes genome using BLAST with characterized enzymes as query. The search identified genes potentially coding for RNA modifying enzymes responsible for most of the found modifications.

Keywords: 23S RNA; RNA methylations; RNA modifications; dihydrouridine; mass spectroscopy; oh5C.

Figure 1.

A secondary structure model of C. sporogenes 23S rRNA (http://www.rna.icmb.utexas.edu). Green overlay shows areas isolated for MS analysis with the names of the complementary oligodeoxynucleotides used for hybridization. The overlapse between BV616 and BV617 and between BV483 and BV608 are indicated by L-shaped symbols. Red overlay shows areas used for hybridizing DNA for primer extension analysis. Modified nucleotides are pointed to by arrows, using E. coli numbering though the 23S rRNA sequence is C. sporogenes. Ec is abbreviation for E. coli and Cs for C. sporogenes.

Figure 2.

MS analyzes of the fragment isolated by the oligodeoxynucleotide BV483 covering position 2480 to 2527. (a) MALDI TOF mass spectrum of RNase T1 digested 23S rRNA sub-fragment. The labelled signals correspond to the 3´-phosphate products, but intense signals corresponding to 2´-3´-cyclic phosphate products (*) also appear approximately 18.0 Da. lower as exemplified in the insert. The table shows m/z values of unmodified RNA and compares to observed values and interpretation of the mass increase; the latter is based on modifications reported in literature, digestion with RNase A and tandem MS data as explained in the main text. (b) MALDI TOF mass spectrum of RNase A digested 23S rRNA sub-fragment. The table indicates m/z values of unmodified RNA and compares to observed values and interpretation of the mass increase; the latter is based on modifications reported in literature, digestion with RNase T1 and tandem MS data as explained in the main text. The insert illustrates m/z signals and isotope patterns leading to the assignment of m²A2503. Unlabelled signals have m/z values corresponding to gas phase-formed dimers of the RNase products around 660 and 1000 m/z. (c) Tandem mass spectrum of the m/z 2237.3 RNase T1 digestion product (position 2496–2502 of 23S rRNA) harbouring 18.0 Da. posttranscriptional modifications. Unambiguous backbone fragment ions, key N-glycosidic bond cleavage products and a few other prominent fragment signals are assigned. The insert indicates how backbone fragment ions contribute to the sequence determination. Fragment ion nomenclature is according to [50].

Figure 2.

(Continued.)

Figure 3.

Tandem MS analyses of subfragments isolated by the oligodeoxynucleotide BV607 covering position 2418 to 2460. (a) Tandem mass spectrum of the m/z 2064.3 RNase A digestion product (position 2444–2449 of 23S rRNA) harbouring 30.0 Da. posttranscriptional modifications. Unambiguous backbone fragment ions, key N-glycosidic bond cleavage products and other prominent fragment signals are assigned. The insert indicates how fragment ions correlate to the sequence. (b) Tandem mass spectrum of the m/z 2289.3 RNase T1 digestion product (position 2448–2454 of 23S rRNA; 2´-3´cyclic phosphate) harbouring 16.0 Da. posttranscriptional modifications. Unambiguous backbone fragment and other prominent fragment signals are assigned. The insert indicates how backbone fragment ions correlate to the sequence.

Figure 4.

Primer extension analysis to pinpoint nucleotides with modifications. The long vertical lines separate E. coli and C. sporogenes samples and the shorter lines separate sequencing and dNTP variation. G, A, U, and C indicate sequence lanes with dideoxynucleotide-sequencing. (a) Extension from primer BV632 (Figure 1). The first four lanes are sequencing of 23S rRNA from E. coli, the next four lanes are sequencing of 23S rRNA from C. sporogenes, then follows five lanes with decreasing dNTP concentrations (0.64, 0.16, 0.08, 0.02, and 0.01mM), see text for discussion. The next five lanes are with decreasing dNTP concentrations on E. coli 23S rRNA (0.5, 0.1, 0.04, 0.02, and 0.004mM) and show a dNTP-dependent stop at U2552, indicating a ribose methylation. The rightmost four lanes show sequencing. (b) Extension from primer BV631 (Figure 1). The first four lanes are sequencing lanes with 23S rRNA from C. sporogenes. There is not clear sequencing close to the primer as is often seen, but there is a complete stop at position 1915 and a partial stop at 1920. The next five lanes show extension on the same RNA with decreasing dNTP concentrations (0.5, 0.1, 0.04, 0.02, and 0.004mM). This shows a dNTP-dependent stop at 1920, indicating a ribose methylation. The last five lanes show sequencing of 23S rRNA from E. coli plus a control (R) without ddNTPs. The absence of stop at C1920 and the clear stop at U1915 are indicated by arrows.