Association of polyadenylation cleavage factor I with U1 snRNP

Abstract

Splicing and polyadenylation factors interact for the control of polyadenylation and the coupling of splicing and polyadenylation. We document an interaction between the U1 snRNP and mammalian polyadenylation cleavage factor I (CF Im), one of several polyadenylation factors needed for the cleavage of the pre-mRNA at the polyadenylation site. Sucrose density gradient centrifugation demonstrated that CF Im separated into two fractions, a light fraction which contained the known CF Im subunits (72, 68, 59, and 25 kD), and a heavy fraction, rich in snRNPs, which contained predominately the 68-and 25-kD CF Im subunits. Using specific antibodies we found that the heavy fraction contains U1 snRNP/CF Im coprecipitable complexes. These complexes were insensitive to RNase treatment, suggesting that the coprecipitation is not due to RNA tethering. In vitro binding experiments show that both the 68-and 25-kD subunits bind to and comigrate with U1 snRNP. In addition, the 25-kD CF Im subunit binds specifically to the 70K protein of U1 snRNP (U1 70K). This binding may account for the CF Im/U1 snRNP interaction. During these studies we found that mAb 2.73 (mAb 2.73), an established U1 70K antibody, efficiently precipitates the bulk of the CF Im from cellular extracts. Because mAb 2.73 has been used in a number of previous studies related to the U1 snRNP and the U1 70K protein, the precipitation of CF Im must be considered in evaluating past and future data based on the use of mAb 2.73.

Serine/Arginine-Rich Proteins Contribute to Negative Regulator of Splicing Element-Stimulated Polyadenylation in Rous Sarcoma Virus

“…Samples were then proteinase K treated, phenol extracted, and ethanol precipitated, and RNA was resolved on a 6% polyacrylamide gel that contains 8 M urea. For U1 snRNP inactivation, HeLa nuclear extract was preincubated for 10 min at 30°C with 100 M 2Ј-O-methyl RNA oligonucleotide U1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] (47) or U7 [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] (48). Polyadenylation reaction mixtures with S100 contained 34% HeLa S100 extract supplemented with 16% HeLa nuclear extract alone or with 1 g purified SR proteins (58).…”

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

“…The domain organization resembles SR proteins involved in pre-mRNA splicing. In fact, CFI68 was shown to copurify with the spliceosome (122,123) and interacts with splicing factors (117,124), suggesting its potential role in coordinating pre-mRNA splicing and 3= processing. The RRM interacts weakly with RNA, but the affinity can be enhanced appreciably by cooperative binding of CFI25 (117).…”

Splicing-independent recruitment of U1 snRNP to a transcription unit in living cells

“…The nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5Ј splice site (Lewis et al, 1996). U1-70K binds the 25-kD cleavage factor I subunit CF-I m (Awasthi and Alwine, 2003). The U1A protein interacts with the 160-kD subunit of the cleavage-polyadenylation specificity factor (CPSF) (Lutz et al, 1996) and CPSF forms a complex with TFIID of the basal transcription machinery (Dantonel et al, 1997).…”