rRNA-like sequences occur in diverse primary transcripts: implications for the control of gene expression

Abstract

Many eukaryotic mRNAs contain sequences that resemble segments of 28S and 18S rRNAs, and these rRNA-like sequences are present in both the sense and antisense orientations. Some are similar to highly conserved regions of the rRNAs, whereas others have sequence similarities to expansion segments. In particular, four 18S rRNA-like sequences are found in several hundred different genes, and the location of these four sequences within the various genes is not random. One of these rRNA-like sequences is preferentially located within protein coding regions immediately upstream of the termination codon of a number of genes. Northern blot analysis of poly(A)+ RNA from different vertebrates (chicken, cattle, rat, mouse, and human) revealed that a large number of discrete RNA molecules hybridize at high stringency to cloned probes prepared from the 28S or 18S rRNA sequences that were found to match those in mRNAs. Inhibition of polymerase II activity, which prevents the synthesis of most mRNAs, abolished most of the hybridization to the rRNA probes. We consider the hypotheses that rRNA-like sequences may have spread throughout eukaryotic genomes and that their presence in primary transcripts may differentially affect gene expression.

Figure 1

Sequence matches to mouse rRNAs based on fasta searches of nucleic acid data bases. Similarities of mRNAs and gene sequences to the mouse rRNA sequence are indicated as lines below the corresponding regions of rRNA. The extent of the homologies is indicated in color. The numbers above the bar representing the rRNA indicate the size of the rRNA in nucleotides. Locations of the 24 probes used for the hybridization analyses (see Fig. 4) are indicated below the bars. The black bars (28S and 18S rRNA) correspond to expansion segments. Note that the 18S rRNA sequence matches are from mouse data base entries only. The shaded bars (5S-1 and -3) are genomic sequences. Hundreds of sequences had similarity to the expansion segment at 28S nt 2654–3279. The region of the α-sarcin domain is indicated by the symbol ∗. The segments that form columns of sequence matches at nt 250–300 (18S-a), nt 679–768 (18S-b), nt 918–930 (18S-c), and nt 1286–1334 (18S-d) were found in more than 1000 mouse data base entries. The extent of these sequence similarities over approximately 1000 matches for 18S-a ranged from 83% over 29 nt to 75% over 24 nt; for 18S-b, they ranged from 80% over 35 nt to 72% over 33 nt; for 18S-c, they ranged from 100% over 13 nt to 100% over 8 nt; and for 18S-d, they ranged from 94% over 49 nt to 60% over 40 nt. In each of these ranges, the shorter match was to a subset of the longer nucleotide sequence.

Figure 2

Location of mouse 18S rRNA-like sequences within and outside of transcription units. One hundred fasta matches to the 18S rRNA sequences indicated were analyzed for the location of the sequence similarity. The total number of matches in 5′ UTRs (5′), coding sequences (C), introns (I), and 3′ UTRs (3′) is indicated by the length of the filled/stippled bar. The relative number of matches identified either in the sense or reverse complementary orientation is indicated by the length in each bar of the stippled or filled regions, respectively. The total number of matches identified in genomic DNA sequences that were not in transcription units (G) is shown by the open bars.

Figure 3

Location of an 18S rRNA sequence upstream of termination codons. The bottom sequence (18S) is the reverse complement of mouse 18S rRNA, nucleotides 1286–1334. Above this sequence are aligned the sequences of Xenopus homeobox XgbX2 mRNA (XGB, accession no. U04867U04867), mouse myosin light chain 2 mRNA (MYO, accession no. M91602M91602), mouse ATP-dependent RNA helicase (HEL, accession no. U46690U46690), mouse lactate dehydrogenase-A4 mRNA (LDL, accession nos. M17516M17516 and M29170M29170), human epithelial membrane protein 2 (EMP, accession no. X94771X94771), and mouse pre-T cell receptor α-type chain precursor mRNA (PTR, accession no. U16958U16958). Matches are represented as vertical bars. The translation termination sequences are boxed. The number 1 in the HEL sequence represents the sequence CG.

Figure 4

Northern analysis with rRNA probes under high stringency conditions. (A) Mouse embryo RNA probed with 28S-1 to 28S-11, 18S-1 to 18S-7, 5S-1 to 5S-3, and 5.8S-1 to 5.8S-2 inserts (see Fig. 1). Lane numbering corresponds to probes. All lanes were loaded with 1 μg of poly(A)⁺ RNA, except for lanes 5.8S-1t and 5.8S-2t, which had 10 μg of total RNA. (B) Northern analysis of RNA from multiple adult mouse tissues with 28S.11 insert probe. Each lane was loaded with 2 μg of poly(A)⁺ RNA from heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. (C) Northern hybridizations using 1 μg of poly(A)⁺ RNA from adult human (lane 1), cow (lane 2), and chicken brain (lane 3); Drosophila embryo (lane 4); and yeast (lane 5). The left, center, and right gel sets were probed with the 28S.2, the 28S-11, and the 18S-1 insert probes, respectively. (D) Northern hybridizations using poly(A)⁺ RNA prepared from cells that were cultured for 2 days in the absence (lane 1) or presence (lane 2) of 50 μg/ml α-amanitin. The positions of the 28S, 18S, 5.8S, and 5S rRNAs are indicated to the left in A. In B and D, the position of the 18S rRNA, and in panel C, the positions of the 28S and 18S rRNAs are shown at the left.