Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.
We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.
Lutz
C S
CS
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104 USA.
Murthy
K G
KG
Schek
N
N
O'Connor
J P
JP
Manley
J L
JL
Alwine
J C
JC
D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011061:Poly A / Q000096:biosynthesis; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors
D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D039221:mRNA Cleavage and Polyadenylation Factors; D011061:Poly A; D005982:Glutathione Transferase; D011062:Polynucleotide Adenylyltransferase