Identification of an activity in B-cell extracts that selectively impairs the formation of an immunoglobulin mu s poly(A) site processing complex.

The immunoglobulin mheavy-chain transcription unit is differentially expressed during B-cell development, producing mRNAs that encode secreted (ms) and membrane-bound (mm) forms of the heavy-chain polypeptide. Whereas the ms mRNA and the mm mRNA are produced in approximately equal abundance in B cells, an increase in the utilization of the ms poly(A) site contributes to the production of the ms mRNA as the predominant form in a plasma cell. Previous experiments have demonstrated a correlation between the formation of a stable complex on a poly(A) site and the relative function of the poly(A) site. We have thus investigated the parameters determining the interaction of these factors with the immunoglobulin poly(A) sites.Assaysofcomplexformationinvolvingthetwoimmunoglobulinpoly(A)sitesbyusingHeLacellactivities revealed the formation of stable complexes with no apparent difference between the ms site and the mm site. In contrast, the ms-specific complex was markedly less stable when a B-cell extract was used. Fractionation of B-cellextractshasrevealedanactivitythatspecificallydestabilizesthe mspolyadenylationcomplex,suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors. The control of gene expression can involve regulated alterations of virtually any step in the process of mRNA biogenesis, including the formation of the 39terminus of an mRNA (21). This process, commonly known as poly(A) site formation, involves a recognition of specific sequence elements in the premRNA followed by endonucleolytic cleavage of the premRNA and then the addition of approximately 200 adenylate residues(16,17,35,37).Therecognitionofessentialsequences and accurate processing of the pre-mRNA can be reproduced in cell-free systems, and this has allowed the identification and purification of cellular components that mediate this event (1, 3, 4, 8, 10, 18, 20, 29‐34). These studies have demonstrated the involvement of several large, multicomponent activities that recognize sequence elements in the RNA through the formation of RNA-protein complexes (9, 12, 28, 38). Although in most cases the processing of an RNA at a poly(A) site is an essential but not a regulated event, there are instances where alterations in poly(A) site selection appears to regulate gene expression (15). Perhaps the best-studied example is found in the immunoglobulin (Ig) heavy-chain transcription unit. During B-cell development, expression of the rearranged heavy-chain locus leads to the production of two distinctmRNAs,oneencodingamembrane-boundformofthe m heavy-chain polypeptide (mm) and a second encoding the secreted form of the heavy chain (ms) (11). These two RNAs are produced by a combination of alternative RNA splicing (19, 24, 27) as well as alternative use of two poly(A) sites (6, 7, 13, 14, 19). In pre-B and mature B cells, approximately equal amounts of the two RNAs are produced, whereas in an antibody-secreting plasma cell, the ms RNA predominates. PreviousfunctionalanalysesofIgheavy-chainRNAprocessing have revealed that the distal mm (or gm) poly(A) site is considerablystrongerthantheproximal ms(or gs)poly(A)site and that the organization of the transcription unit critically