Complexity of poly(A+) and poly(A-) polysomal RNA in mouse liver and cultured mouse fibroblasts.

RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.     

Apolipoprotein B mRNA sequences 3'' of the editing site are necessary and sufficient for editing and editosome assembly.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.     

Frequency distribution of messenger sequences within polysomal mRNA and nuclear RNA from rat liver.

DNA complementary to polysomal poly(A)-containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes. 1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10,000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome. 2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm. 3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.     

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.     

Direct demonstration of a complementarity between mRNA and double-stranded sequences of pre-mRNA

The total poly(A)-containing mRNA from mouse liver or Ehrlich ascites carcinoma cells was annealed with denatured ds RNA prepared from heavy nuclear ³H-labeled pre-mRNA of the same tissue. The hybrids formed were detected by binding of complexes to poly(U)-Sepharose columns through the poly(A) of mRNA. With this technique, about 30% of labeled ds RNA was bound to poly(U)-Sepharose after annealing it with an mRNA excess. The proportion of hybrid material detected by RNase treatment was two to three times lower than that obtained by poly(U)-Sepharose binding. The length of the RNase-stable acid precipitable hybrid material consisted of heterogeneous sequences of 10–100 nucleotides long when cytoplasmic, and 10–60 nucleotides long when polysomal mRNA was used in the hybridization reaction. The results obtained show that at least some of the mRNA molecules contain sequences complementary to one of the branches of the pre-mRNA hairpins. These results are compatible with the idea that the hairpin-like sequences in pre-mRNA are localized between mRNA and the non-informative part of the precursor molecule.     

Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.

We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.     

Accumulation of polyadenylated mRNA during liver regeneration.

Cytoplasmic and polysomal polyadenylated mRNA [poly(A)+-mRNA] increased by 120% prior to the onset of DNA synthesis during the regeneration of rat liver following partial hepatectomy. Despite this large change in cytoplasmic mRNA and an approximately 50% increase in total nuclear RNA, the amount of polyadenylated nuclear RNA increased by only 15--20% during this time. Neither the average size of nuclear or of cytoplasmic polyadenylated mRNA nor the length of their poly(adenylic acid) [poly(A)] tracts changed during liver regeneration. Polysomal poly-(A)+-mRNA increased proportionately more and at a faster rate than rRNA during the first day following partial hepatectomy. Normal livers contained a substantial proportion of cytoplasmic poly(A)+-mRNA not associated with polysomes but this proportion was not altered in 3-h regenerating liver. Thus, in regenerating liver, most preexisting cytoplasmic mRNA does not appear to be recruited into polysomes prior to the substantial increase in the amount of cytoplasmic poly(A)+-mRNA.     

Identity elements used in export of mRNAs

Different classes of RNA are exported from the nucleus by distinct factors. We demonstrate that U1 snRNA is exported like an mRNA on insertion of a pre-mRNA intron or either sense or antisense mRNA exon sequences. mRNA-specific factors are recruited onto the spliced or elongated U1 RNA whereas U snRNA-specific factors are not, suggesting that an unstructured region of sufficient length in an RNA acts as a dominant determinant of mRNA identity. After export, spliced U1 RNA undergoes cytoplasmic maturation but is not reimported into the nucleus. These data provide insight into mechanisms for discrimination of different classes of nuclear RNA and demonstrate that two RNAs of identical sequence can have distinct cytoplasmic fates depending on their mode of export.