FIG. 2.

The lamin B2 USE is required for efficient cleavage and poly(A) addition in vitro. (A) Cleavage assays were performed in nuclear extract using wt, Sp, USEmt, or ISEmt RNAs. The positions of the input RNAs (solid- and -open box) and the 5′ cleavage products (solid box) are indicated to the right of the gel. Note that for the Sp RNA (lanes 6 to 9), both the input and the 5′ fragment are slightly longer than those for the other RNAs. (B) Poly(A) addition reactions in nuclear extract using the pre-wt, pre-Sp, pre-mt, and D1 RNAs. [The D1 RNA has no poly(A) signal sequences.] The positions of the input and poly(A)⁺ RNAs are indicated. (C) Poly(A) addition is qualitatively different for the pre-wt and pre-Sp substrates. The graphs represent quantitation of lanes 5 and 9 of panel B—the 60-min time points. Arrows indicate the positions of the input RNA, and brackets show the extent of the heterogeneous poly(A)⁺ band.