Complex alternative RNA processing generates an unexpected diversity of poly(A) polymerase isoforms.
Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have previously been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, we isolated the mouse PAP gene and an intronless pseudogene from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which we call PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive. We discuss the possible significance of this complex expression pattern.
Zhao
W
W
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Manley
J L
JL
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002417:Cattle; D017931:DNA Primers; D005091:Exons; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012694:Serine; D018411:Spodoptera; D013912:Threonine; D014162:Transfection; D014981:Xenopus
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D011062:Polynucleotide Adenylyltransferase