Sensitivity to UV radiation of small nuclear RNA synthesis in mammalian cells.

Abstract

It was demonstrated previously that the synthesis of small nuclear RNA (snRNA) species Ut and U2 in human cells is very sensitive to UV radiation. In the present work, the UV sensitivity of U3, U4, and U5 snRNA synthesis is shown to be also high. The synthesis of Ut, U2, U3, U4, and U5 snRNAs progressively decreased during the first 2 h after UV irradiation (this was not observed in polyadenylated RNA) and had not returned to normal rates 6 h after UV exposure. In contrast, the restoration of 5.8S rRNA synthesis began immediately after UV irradiation and was essentially complete 6 h later. A small fraction of Ut and U5 (and possibly U2 and U3) snRNA synthesis remained unaffected by high UV doses, when cell radiolabeling began 10 min after UV irradiation. The present data suggest that a factor other than the level of pyrimidine dimers in DNA (possibly, steps in the post-irradiation DNA repair process) plays an important role in the mechanism of UV-induced inhibition of U1-U5 snRNA synthesis.UV radiation causes random formation of pyrimidine dimers in DNA, which result in premature termination of transcription (23). This effect has been used to determine the length of transcription units, since the probability of causing a UV lesion within a transcription unit is directly proportional to its length. Human Ut and U2 small nuclear RNA (snRNA) synthesis is very sensitive to UV radiation (4), suggesting that the UV target sizes of Ut and U2 snRNAs are much larger than those of the known precursors to these snRNAs (5). In contrast, the synthesis of small RNAs whose primary transcripts are not much longer than those of the mature species, like 4S and 5S RNA, is not affected by the same dose range of UV radiation (4, 8). Mammalian snRNA species Ul, U2, U3, U4, and U5 share several common properties, including having N2,N2,7-trimethylguanosinecapped structures at their 5' ends (3) and possibly being synthesized by RNA polymerase II (5, 7, 9, 12, 21, 22; S. C. Chandrasekharappa, J. H. Smith, and G. L. Eliceiri, J. Cell. Physiol., in press). The original goal of the present work was to determine whether U3, U4, and U5 snRNA synthesis is also sensitive to UV radiation; our results show that it is. Ul through U5 snRNA synthesis decreases during the first 2 h after UV irradiation and is not restored to normal levels for at least 6 h after UV exposure. These results t Present address: Cancer Research Laboratory, University of Western Ontario, London, Ontario, Canada N6A SB7.suggest that a factor other than the pyrimidine dimer content of DNA is important in the UV inhibition of U1-U5 snRNA synthesis.MATERIALS AND METHODS KB or HeLa (human) cells, grown in spinner culture, were exposed to UV light as described earlier (at 15 cm from a GE G2578 lamp) (4). After specified times in spinner culture at 37°C, the cells were labeled with [3H]uridine (40 p.Ci/ml) for 40 min, except for the experiment in Fig. 1 and 2 where labeling was for 80 min. The radioactive medium was removed, and the cells were incubated for 2 h in fresh medium su...

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

“…Several reports suggest that there may be precursors to the snRNAs at the 5' end (10,12). In a DNA-dependent system from HeLa cells, the sole transcript formed is initiated 183 bases 5' to the first base of the mature RNA (29).…”

“…These genes have extensive homology in the 5' flanking region extending 70 bases 5' from base 1 of the RNA (23). The Ul genes also differ from other RNA polymerase II transcripts in that they show UV sensitivity very different from that predicted from the size of the RNA product (10,12). Based on this UV sensitivity, it has been proposed that these RNAs are synthesized as part of a large transcript.…”

Effect of UV light on small nuclear RNA synthesis: increased inhibition during postirradiation cell incubation.

“…The term early inhibition will be used here for the UV light-induced suppression of snRNA biosynthesis that is easily seen after relatively high UV doses and very short postirradiation cell incubations (for example, about 290 J/m2 and no more than 10 min, respectively). Second, a small fraction of Ul, U2, U3, and U5 snRNA biosynthesis remains unaffected at high UV doses when cell radiolabeling begins soon (within 10 min) after irradiation (6). The presence of a UV lightresistant subpopulation of RNA synthesis has not been observed in any other RNA species whose synthesis is inhibited by UV radiation.…”

“…There are three unexpected features about the effect of UV radiation on the biosynthesis of small nuclear RNA (snRNA) species Ul, U2, U3, U4, and U5. First, although Ul through U5 snRNAs and their known precursors are very short (1,5), their biosynthesis is very sensitive to UV light (3,6). In contrast, for all other RNA species tested there is a strict correlation between the length of the transcription unit and the UV light sensitivity of its expression (24).…”

The p53 Tumor Suppressor Protein Represses Human snRNA Gene Transcription by RNA Polymerases II and III Independently of Sequence-Specific DNA Binding

“…A role for p53 in this process is suggested from two sources. Firstly, in response to UV light treatment, human U1 and U2 snRNA genes exhibit a delayed and prolonged reduction in transcription by RNA polymerase II (14,47,48). In part, this reduction may be attributable to increased hyperphosphorylation of the carboxy-terminal domain of the RNA polymerase II largest subunit in response to UV light (27).…”