Organization and transient expression of the gene for human U11 snRNA

Abstract

The nucleotide sequence of U11 small nuclear RNA, a minor U RNA from HeLa cells, was determined. Computer analysis of the sequence (135 residues) predicts two strong hairpin loops which are separated by seventeen nucleotides containing an Sm binding site (AAUUUUUUGG). A synthetic gene was constructed in which the coding region of U11 RNA is under the control of a T7 promoter. This vector can be used to produce U11 RNA in vitro. Southern hybridization and PCR analysis of HeLa genomic DNA suggest that U11 RNA is encoded by a single copy gene, and that at least three genomic regions could be U11 RNA pseudogenes. A HeLa genomic copy of a U11 gene was isolated by inverted PCR. This gene contains the U11 RNA coding sequence and several sequence elements unique for the U RNA genes. These include a Distal Sequence Element (DSE, ATTTGCATA) present between positions -215 and -223 relative to the start of transcription; a Proximal Sequence Element (PSE, TTCACCTTTACCAAAAATG) located between positions -43 and -63; and a 3' box (GTTAGGCGAAATATTA) between positions + 150 and + 166. Transfection of HeLa cells with this gene revealed that it is functioning in vivo and can produce U11 RNA.

Figure 1

Sequence of U11 RNA. The secondary structure was predicted with the GCG “RNA Fold” program (Devereux, 1990). The Sm binding site (consensus RAU_[3–6]GR) is boxed, and the TMG cap is indicated as m₃G.

Figure 2

Southern analysis of human placenta DNA. Human placenta DNA was digested with EcoRI, BamHI, and NcoI; separated by 1% agarose gel electrophoresis; and transfected to Zetaprobe membrane. The filter was incubated with Cs32 riboprobe (containing U11 RNA preceded by 3 guanosine residues).

Figure 3

The human U11 gene. HeLa DNA digested with DdeI and HaeIII was used for cloning of the human U11 gene by inverted PCR. Shown here is the sequence of this locus between the two HaeIII sites. Thick underline: the UII coding sequence (position +1 to +135), the distal sequence element (−223 to −215), the proximal sequence element (−62 to −41), and the 3′ box (+150 to +165). Further indicated are the Alu sequence (−855 to −717, thin underline); two indirect repeats and two direct repeats (arrows); and several restriction sites (HaeIII, GG′CC; DdeI, C′TGAG; EcoRI, G′AATTC; PvuII, CAG′CTG).

Figure 4

Comparison of the regulatory elements of human U RNA genes. The schematic (top) shows the general organization of human U RNA genes, including DSE, PSE, and 3′ box (Dahlberg and Lund, 1988). Below, these three transcription elements for each U snRNA gene are depicted in more detail. The consensus of each is shown at the bottom (R = A or G, Y = C or T, S = C or G and W = A or T). Residues in the transcription elements of the U snRNA genes that do not fit the consensus are shown in small print. The positions between which the consensus of each element is found are indicated by the numerals below (exception is the DSE of U4b, which is found around position +155). The positions of the three elements in the U11 gene are also indicated. In this figure, the position of the 3′ box is relative to the stop of transcription. Note: the U6 RNA gene is transcribed by RNA polymerase III and has a stretch of five T residues as terminator. The DSE elements of U4c, U6, and U11 are presented here in the opposite orientation of how they occur in their respective genes (see Murphy et al., 1982; Htun et al., 1985; Yuan et al., 1989; Bark et al., 1986; Kunkel et al., 1986).

Figure 5

Transient expression of the U11 gene. HeLa cells were transfected by calcium phosphate co precipitation. Twelve hours after transfection the medium was renewed, and total cellular RNA was isolated 30 hours later, separated by denaturing PAGE and analyzed by Northern hybridization. Cells were transfected with the following constructs: lane 1, 1.5 Hg pU11a and 1.5 μg pSVEori⁻; lane 2, 3 μg pU11a and 3 μg pSVEori⁻; lane 3, 1.5 μg pU11a.ori and 1.5 μg pSVEori⁻; lane 4, 3 μg pU11a.ori and 3 μg pSVEori-; lane 5, 1.5 μg pU2/−247 and 1.5 μg pSVEori⁻. A. Northern filter probed with Cs32 riboprobe (high stringency conditions). B. the same filter probed under low stringency conditions with pU2/t3 (Cs32 riboprobe was not removed). Filters were exposed to film for 16 hours.