Variability of polyadenylation sites in mRNAs from human fetal liver

cDNA libraries of human fetal liver were constructed in pBR322 and λgt10 vectors. The libraries were screened for liver-specific clones by differential hybridization. This procedure revealed 25 and 32 liver-specific clones in plasmid and phage libraries, respectively. The majority of these clones were represented with serum albumin, fetal ^Gγ-globin and ^Aγ-globin cDNA inserts. Three types of 3′-non-coding region were found in 5 sequenced albumin cDNAs. In one type mRNA the distance between the AATAAA signal and polyadenylation site was 15 nucleotides, in 2 other types this distance was 10 and 6 nucleotides. The polyadenylation site in the ^Gγ-globin cDNA was located 2 nucleotides further from AATAAA signal, while in the ^Aγ-globin cDNA it was 2 nucleotides closer to the signal as compared with the results published previously.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Gene structure of human CD59 and demonstration that discrete mRNAs are generated by alternative polyadenylation.

We have isolated the CD59 gene from human genomic libraries. The gene is distributed over more than 27 x 10(3) base-pairs and consists of one 5'-untranslated exon and three coding exons. The gene structure is similar to that of mouse Ly-6 with the exception of the larger size of CD59 introns. Northern blot analysis using six different probes located in the 3'-region of the gene shows that more than four different CD59 mRNA molecules are generated by alternative polyadenylation. Three of these polyadenylation sites were predicted from previously published cDNA sequences. We have isolated a fourth from Jurkat poly(A)+ RNA by the procedure of rapid amplification of cDNA ends. Alternative polyadenylation may be due to the RNA secondary structure around the typical polyadenylation signal, AAUAAA.     

Properties of human liver cytosolic aspartate aminotransferase mRNAs generated by alternative polyadenylation site selection

Human cytosolic aspartate aminotransferase (cAspAT) cDNA clones have been isolated from an adult human liver cDNA library. Among the clones, two cDNAs of 1550 and 1950 base pairs, respectively, have been characterized. These two cDNAs differ only in the lengths of their 3' noncoding regions and by the presence of one or two putative polyadenylation signals AATAAA. Northern blot analysis revealed two different mRNAs of 2.1 and 1.8 kbp in several human tissues, whereas Southern blot analysis suggested the existence of a single gene for the human cAspAT. The two mRNA species result from the alternative use of two polyadenylation signals. In the liver, the relative ratio of these mRNAs varies among different species and, in humans at least, during development. The properties of the two mRNAs were compared. The half-lives of the 2.1 and 1.8 kbp mRNAs, in the HepG2 cell line, are 8 and 12 h, respectively. The two mRNAs have similar and rather short poly(A) tracts of 20-50 nucleotides. Both mRNAs are capable of directing the in vitro synthesis of the cAspAT protein. We conclude that both the 2.1 and 1.8 kbp cAspAT mRNAs are functional and exhibit similar properties.     

A single mouse alpha-amylase gene specifies two different tissue-specific mRNAs

The alpha-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5' non-translated sequences: the 5' terminal 158 nucleotides of the major liver alpha-amylase mRNA are unrelated to the 5' terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5'terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver alpha-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5' terminal 50 nucleotides of the salivary gland alpha-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver alpha-amylase mRNAs are transcribed from the same gene (Amy1A). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amy1A. Rather, these data indicate that the salivary gland and liver alpha-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.     

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3-untranslated regions (3-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3 non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event ocurring in this intron.     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.