Multiple factors are required for poly(A) addition to a mRNA 3' end.

Abstract

Polyadenylation of pre-mRNAs in the nucleus involves a specific endonucleolytic cleavage, followed by the addition of -200 adenylic acid residues. We have assayed HeLa nuclear extracts for the activity that catalyzes the poly(A) addition reaction. The authenticity of the in vitro assay was indicated by the observation that the poly(A) addition reaction requires an AAUAAA element near the 3' terminus of the substrate and that the poly (A) tract added in vitro is -200 nucleotides in length. We have fractionated nuclear extracts in order to define components involved in specific poly(A) addition. No single fraction from DEAE-Sephacel chromatography of a HeLa nuclear extract possessed the specific poly{A) addition activity. However, if the various fractions were recombined, activity was restored, indicating the presence of multiple components. Further fractionation revealed the presence of at least two factors necessary for the poly(A) addition reaction. The reconstituted system retains the characteristics and specificity seen in the crude extract. Additional purification of one of the factors strongly suggests it to be a previously characterized poly(A) polymerase which, when assayed in the absence of the other factor, can add AMP to an RNA terminus but without specificity. Thus, the other component of the reaction may provide specificity to the process. In contrast to the 3' cleavage reaction, the poly{A) addition machinery does not possess an essential RNA component, as assayed by micrococcal nuclease digestion, nor do anti-Sm sera inhibit the reaction. Thus, the total process of formation of a polyadenylated mRNA 3' end is complex and requires the concerted action of distinct nuclear components.

The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase.

“…The fraction used in these experiments eluted at approximately 300 mM KCl. Our data directly demonstrate that the enzyme responsible for polyadenylation of mRNAs is the same as the poly(A) polymerases first purified 15 years ago and is consistent with the observations of others on the HeLa enzyme (2,11,17,18). In retrospect poly(A) polymerase was the first enzyme involved in nuclear mRNA processing to have been identified and characterized.…”

CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition.

“…The DNAs that served as templates for the in vitro transcription of the precleaved pre-mRNAs L3, and AAC were digested with AccI (McDevitt et al 1988). The analogous templates for Ause/CPS, Ause/hB, d~use/~C, and Ause/ABC were produced by PCR, because of the presence of both an XhoI and an AccI site within the A segment of the Ause linker substitution {NXS 9571-9588; Valsamakis et al 1991).…”

“…Oligonucleotides containing the appropriate sequences were utilized along with unique restriction sites to generate exact HIV/L3 junctions. The C/D segment junctions were formed by digestion with XhoI (HIV-1) or AccI (L3), which encompasses the cleavage sites of the HIV-1 (see Gilmartin et al 1992) and L3 (see McDevitt et al 1988) poly(A) sites, respectively, followed by treatment with Klenow polymerase and DNA ligase.…”

Four factors are required for 3'-end cleavage of pre-mRNAs.

“…On the other hand, the PAP activity is also required to cleave all other pre-RNAs tested so far. Using different fractionation methods, it has been shown that multiple factors are required for both cleavage and polyadenylation reactions McDevitt et al 1988). Christofori and Keller (1988) have recently demon-Cold Spring Harbor Laboratory Press on May 11, 2018 -Published by genesdev.cshlp.org Downloaded from strated that three factors are required for cleavage of both SV40 late and Ad2 L3 pre-RNAs.…”