FIGURE 5.

Purification of U1A-associated proteins. A, protein composition of the U1A complex. Purification was performed using ∼2 × 10¹¹ cells, as described under “Experimental Procedures.” The purified proteins were separated on a 12% SDS-polyacrylamide gel and stained with silver. The molecular mass markers are indicated. B, specificity of snRNA selection using the U1A-tagged protein. Extract was prepared from cells as described (13). RNA was extracted from IgG-agarose beads and subjected to primer extension with the different probes (supplemental S-1). RNA was prepared from an aliquot of cells before selection (Total), and RNA from the beads (Final). Only an aliquot (2%) of the sample was analyzed from the total RNA, whereas the entire sample from the beads was analyzed. C, U1A functions in polyadenylation. Panel a, changes in poly(A) tail during U1A silencing. Cell expressing silencing constructs for either PAP,⁴ U1 70 kDa, and U1A were silenced. RNA was prepared from uninduced cells (−) or after 2 days of silencing (+). RNA was splint-labeled, digested with RNase A and T1, and the labeled tails were separated on a 15% denaturing gel next to a pBR322 DNA-MspI digest. The level of U3 snoRNA was used to determine the amount of RNA in each sample. Panel b, quantitation of the poly(A) tail length. The phosphorimages were scanned, and the intensity of the signals was measured and plotted. The black and gray histograms represent the tail length distributions of uninduced and induced cells, respectively. D, fractionation of U1 snRNP-associated proteins. Whole cell extracts were prepared from 5 × 10⁸ cells and layered on continuous 10–30% (w/v) sucrose gradient in buffer A containing 150 mm KCl. Gradients were centrifuged at 4 °C for 3 h at 35,000 rpm in a Beckman SW41 rotor. S values were determined using the following standards: 30 S, 50 S, and 70 S E. coli ribosomes and the enzyme, catalase (10 S). Following centrifugation, 24 fractions were collected, and RNA and protein samples were prepared after ethanol precipitation. The proteins were subjected to Western analysis with anti-U1 70-kDa antibodies that recognize the PTP-tagged U1A and the U1 70 kDa. U1 snRNA level was determined by primer extension.