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

Abstract

Nuclei from sea urchin blastula embryos synthesize a variety of small RNAs, one of which has identical mobility with sea urchin Ul RNA. This RNA is synthesized by RNA polymerase II and, in a hybridizationselection experiment, was selected by the cloned sea urchin Ul gene. The Ul RNA was initiated with ATP, but not GIP, in isolated nuclei with P-S-and y-S-ribonucleotide triphosphates as substrates. The Ul RNA containing thiophosphate at the 5' end was not capped but accumulated as an uncapped transcript from which the thiophosphate could be removed with calf intestinal phosphatase.

Structure and expression of a second sea urchin U1 RNA gene repeat

“…Transcription of the Ul RNA gene in isolated nuclei Nuclei were prepared from blastula embryos just after hatching as described by Morris and Marzluff (22). Nuclei were incubated in the presence of o(-32P04 CTP for 30 minutes at 250 and RNA was prepared as previously described (22). The RNA was hybridized with the single-stranded M13 DNA probes from both pLvUl.1 and pLvU1.2 immobilized on nitrocellulose.…”

“…The primer was synthesized using an Applied Biosystems DNA Synthesizer and was a generous gift of Dr. Don Sittman. Transcription of the Ul RNA gene in isolated nuclei Nuclei were prepared from blastula embryos just after hatching as described by Morris and Marzluff (22). Nuclei were incubated in the presence of o(-32P04 CTP for 30 minutes at 250 and RNA was prepared as previously described (22).…”

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

“…While cell-free systems have been described which transcribe most other genes accurately, the cell-free systems currently available from mammalian cells do not accurately transcribe the Ul or U2 genes (28,39). We have previously described the development of both nuclear (25) and DNA-dependent (26) systems from sea urchin embryos which transcribe the sea urchin Ul RNA gene accurately.…”

“…There is a sequence about 200 nucleotides 5' to the gene which is essential for transcription in vivo (19) and on injection of the genes into Xenopus oocytes (29,33,39). When the genes are used as a template in a DNA-dependent system, there is transcription which initiates about 25 bases downstream from this upstream promoter element (as if it were functioning like a TATAA box in this system) but no transcription which initiates at the proper start site (28). The promoters of the sea urchin U RNA genes are not utilized in Xenopus oocytes (37), unlike the promoters of other sea urchin genes transcribed by RNA polymerase II.…”

“…These RNAs are encoded by multiple genes and contain an unusual 5' cap structure, 2,2,7 trimethyl guanosine (3). These RNAs are synthesized by RNA polymerase II, as measured by the sensitivity of their synthesis to low concentrations of aamanitin (8,25,33). An exception is U6 RNA, which lacks the cap structure found at the 5' ends of other U RNAs and is believed to be transcribed by RNA polymerase III (13,32).…”