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. 2002 May 28;99(11):7542-7.
doi: 10.1073/pnas.112063799.

Noncoding RNA genes identified in AT-rich hyperthermophiles

Robert J Klein  1 , Ziva Misulovin, Sean R Eddy
Affiliations

Noncoding RNA genes identified in AT-rich hyperthermophiles

Robert J Klein et al. Proc Natl Acad Sci U S A. .

Abstract

Noncoding RNA (ncRNA) genes that produce functional RNAs instead of encoding proteins seem to be somewhat more prevalent than previously thought. However, estimating their number and importance is difficult because systematic identification of ncRNA genes remains challenging. Here, we exploit a strong, surprising DNA composition bias in genomes of some hyperthermophilic organisms: simply screening for GC-rich regions in the AT-rich Methanococcus jannaschii and Pyrococcus furiosus genomes efficiently detects both known and new RNA genes with a high degree of secondary structure. A separate screen based on comparative analysis also successfully identifies noncoding RNA genes in P. furiosus. Nine of the 30 new candidate genes predicted by these screens have been verified to produce discrete, apparently noncoding transcripts with sizes ranging from 97 to 277 nucleotides.

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Figures

Figure 1
Figure 1
GC content as a basis for finding ncRNA genes. (A) GC content of whole genomes vs. optimal growth temperature. In this and subsequent images, the large square represents M. jannaschii, the up triangle P. abyssi, the down triangle P. horikoshii, and the diamond P. furiosus. (B) GC content of tRNA genes vs. optimal growth temperature. (C) Difference in tRNA and genomic GC content vs. optimal growth temperature. (D) GC content of a 1-kb region of the M. jannaschii genome containing a tRNA gene calculated in a 100-bp sliding window.
Figure 2
Figure 2
Northern blots of novel ncRNAs. Each pair of blots represents probing with oligonucleotides for RNA on the + or − strand, respectively. On each blot, the leftmost lane is a 100-bp ladder, the center lane is the RNA sample, and the rightmost lane is a 25-bp ladder.
Figure 3
Figure 3
The genomic context of each novel ncRNA gene. The left-hand column gives the candidate name from the screen. In the center is an independently scaled schematic of the genomic locus. The black arrow represents the longest copy we could find of the ncRNA gene, whereas the gray arrows represent 50 nucleotides of the flanking annotated genes. The numbers above the lines and below the arrows indicate the maximal length of the ncRNA gene as determined by 5′ and 3′ RACE. The other two numbers above the lines are the distances between the gene and its flanking genes, where a number in parentheses indicates the length of the overlap between the two genes. The numbers below the lines represent the start and stop coordinates of the gene. The right-hand column contains the GenBank accession no. for the cDNA sequence.
Figure 4
Figure 4
snoRNA sR9 with the additional stem-loop structure. (A) Predicted secondary structure folding for sR9, with covarying bases in the stem structure noted. The annotations for the C, C′, D, and D′ boxes come from ref. . (B) Northern blot probed with an oligonucleotide complementary to the 2′-O-methyl guide region.
Figure 5
Figure 5
hgcC and its homologues. (A) Northern blot showing expression of homologues of hgcC in M. jannaschii and P. furiosus. (B) Genomic context of hhcA, as in Fig. 3. (C) Genomic context of hhcB, as in Fig. 3, except the full length of ORF PF1918 is included. The dashed line indicates a gap not drawn to scale. The shaded region is not present in the syntenic regions of P. abyssi and P. horikoshii.
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