TY - JOUR
T1 - Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences
AU - Hart, Ronald P.
AU - McDevitt, Michael A.
AU - Nevins, Joseph R.
N1 - Funding Information:
We thank Wai Wong for his superb technical assistance with these experiments. We also wish to thank Peter Model for his assistance with the synthesis of oligonucleotides and the lab of R. Roeder for the HeLa nuclear extract. R. P H. was supported by a National Institutes of Health postdoctoral fellowship; M. A. M. was supported by the Medical Scientist Training Program; and J. R. N. is the recipient of a Research Career Development Award (CAO0866). This work was supported by a grant from the American Cancer Society (MV141B).
PY - 1985/12
Y1 - 1985/12
N2 - Efficient utilization of the early SV40 poly(A) site in vivo requires sequences between 5 bp and 18 bp downstream of the cleavage site. We have used a HeLa nuclear extract to examine the sequence requirements for in vitro cleavage. DNA segments containing the SV40 poly(A) site were cloned into an SP6 vector. SP6 RNAs, accurately cleaved and polyadenylated, were detected by primer extension. Cleavage was enhanced by the presence of a cap on the primary transcript, and was inhibited by the addition of 10 μM 7meGpppG. In close agreement with the in vivo results, efficient processing at the poly(A) site in vitro required the specific downstream sequences in the SP6 RNA transcript. These experiments indicate that the sequence in the RNA precursor downstream of the cleavage site, shown to be important for efficient processing in vivo, is recognized in vitro.
AB - Efficient utilization of the early SV40 poly(A) site in vivo requires sequences between 5 bp and 18 bp downstream of the cleavage site. We have used a HeLa nuclear extract to examine the sequence requirements for in vitro cleavage. DNA segments containing the SV40 poly(A) site were cloned into an SP6 vector. SP6 RNAs, accurately cleaved and polyadenylated, were detected by primer extension. Cleavage was enhanced by the presence of a cap on the primary transcript, and was inhibited by the addition of 10 μM 7meGpppG. In close agreement with the in vivo results, efficient processing at the poly(A) site in vitro required the specific downstream sequences in the SP6 RNA transcript. These experiments indicate that the sequence in the RNA precursor downstream of the cleavage site, shown to be important for efficient processing in vivo, is recognized in vitro.
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U2 - 10.1016/0092-8674(85)90240-5
DO - 10.1016/0092-8674(85)90240-5
M3 - Article
C2 - 2866847
AN - SCOPUS:0022332463
SN - 0092-8674
VL - 43
SP - 677
EP - 683
JO - Cell
JF - Cell
IS - 3 PART 2
ER -