The initial work demonstrating the in vitro 3' processing of exogenous pre-mRNA (Moore and Sharp, 1985) was carried out in a HeLa cell nuclear extract originally developed by Dignam et al. (1985) for the analysis of RNA polymerase II transcription initiation. Subsequent modifications of the Dignam protocol, developed in the Keller lab (Ruegsegger et al., 1996), have yielded superior extracts active for pre-mRNA 3' processing. The Keller nuclear extract may be used directly for 3' processing assays or may be dialyzed into an appropriate buffer prior to use. Dialysis, however, consistently results in a loss of processing activity on the order of approximately twofold. The protocols presented below address the use of both dialyzed and undialyzed nuclear extracts. The extracts are quick frozen in liquid N 2 and stored at — 80°C. Freeze-thawing does not appear to have a large impact on processing activity, but in principle, it should be avoided whenever possible.
Two functional 3' processing assays may be performed in a HeLa cell nuclear extract: (1) endonucleolytic cleavage and (2) poly(A) addition. As discussed below, the use of ATP analogs allows for the independent analysis of these two reactions. Although it is possible to both cleave and polyadenylate a pre-mRNA substrate in vitro, such coupled reactions are generally avoided due to the difficulty encountered in the interpretation of the results. Poly(A) addition is not obligatorily coupled to cleavage of the RNA substrate and PAP has been found to be capable of adding a poly(A) tail to a 3'-OH located over 400 nucleotides downstream of the AAUAAA hexamer (Manley et al., 1985). Since most pre-mRNA substrates used for in vitro 3' processing contain only ~50 to 100 nucleotides downstream of the hexamer, these substrates may be either cleaved and polyadenylated or polyadeny-lated at their 3' termini in the absence of cleavage. When the RNAs are displayed on a denaturing polyacrylamide gel, it is not possible to distinguish the two types of products within the heterogeneous collection of polyadenylated RNAs. Two basic types of RNA substrates are therefore used for the analysis of 3' processing: (1) cleavage substrates and (2) poly(A) addition substrates. Cleavage substrates contain both the AAUAAA hexamer and a downstream element, while poly(A) addition substrates ("pre-cleaved" RNAs) contain only a hexamer and extend to the cleavage site. In general, short RNA substrates (<200 nucleotides) are preferred for in vitro 3' processing. Increasing the size of the pre-mRNA increases the probability of the formation of inactive RNA conformers.
Pre-mRNAs are usually synthesized by phage RNA polymerases SP6 or T7. The DNA templates may consist of linearized plasmid DNA, polymerase chain reaction (PCR) product, or annealed oligos containing a phage RNA polymerase promoter. Since the 5' cap both stabilizes RNAs and enhances 3' processing in nuclear extracts (Gilmartin et al., 1988; Cooke and Alwine, 1996), the pre-mRNAs are synthesized in the presence of m7GpppG. The RNA templates may be either uniformly labeled by transcription in the presence of an [a-32P]NTP or 3' endlabeled with [a-32P]pCp (cytidine 3',5'-bis(phosphate),[5'-32P]) and T4 RNA ligase. Alternative 3' end-labeling techniques have been developed by Lingner and Keller (1993) and by Huang and Szostak (1996). Since the 3' cleavage products are usually at least partially degraded in nuclear extract, 3' end-labeling allows for their positive identification.
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