Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-mRNA Processing In Vivo

Abstract

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. However, not all introns can provide this function. For example, transcripts synthesized from a variant of the human beta-globin gene lacking its second intervening sequence (IVS2), yet retaining its first intervening sequence (IVS1), exhibit multiple defects in mRNA biogenesis. To investigate why, we transfected into monkey cells plasmids containing the human beta-globin gene and variants of it altered in (i) IVS1, (ii) the 3'-terminal exon, and (iii) the polyadenylation signal. The beta-globin RNAs accumulated in these cells were analyzed by quantitative S1 nuclease mapping for nuclear accumulation, intron excision, polyadenylation and cytoplasmic accumulation. We found that the 3' splicing signal of IVS1, with multiple purines interrupting its polypyrimidine tract, could efficiently function as an internal 3' splicing signal; however, it could not efficiently function as the 3'-terminal 3' splicing signal for any of these steps in intron-dependent mRNA biogenesis unless (i) its polypyrimidine tract was made uninterrupted in pyrimidines, or (ii) specific sequences were deleted from the 3'-terminal exon. We conclude that whether an intron can provide the function necessary for efficient processing of intron-dependent pre-mRNA is dependent upon the ability of its 3' splicing signal to define the 3'-terminal exon. On the practical side, this finding means one needs to consider both the sequence and location of the intron to be included in an intron-dependent gene to obtain efficient expression in vivo.

Binding of hnRNP L to the Pre-mRNA Processing Enhancer of the Herpes Simplex Virus Thymidine Kinase Gene Enhances both Polyadenylation and Nucleocytoplasmic Export of Intronless mRNAs

“…␤-Globin primary transcripts lacking both of ␤-globin's introns are defective in RNA stability, proper 3Ј end formation, and nucleocytoplasmic export (3,5,33,46,50) (Fig. 1).…”

“…Plasmids p␤-␤1(ϩ)2(ϩ), p␤-␤1(Ϫ)2(Ϫ), p␤-TK-␤1(Ϫ)2(Ϫ), and p␤-1ϫTK119-␤1(Ϫ)2(Ϫ) have been described previously (32,33,50). They contain the nt Ϫ812 to ϩ2206 region of the human ␤-globin gene relative to the transcription initiation site with (ϩ) or without (Ϫ) ␤-globin's two introns in a pBR322-based cloning vector containing a simian virus 40 (SV40) origin of DNA replication (Fig.…”

“…RNA was purified using TRIzol reagent following the manufacturer's suggestions (Gibco). The relative amounts of globin-like RNA accumulated in the nuclear and cytoplasmic fractions were determined by quantitative S1 nuclease mapping as described previously (32,33) and quantified with a PhosphorImager and ImageQuant software. Cellular ␤-actin RNA served as an internal control for recovery of the RNA samples (32,33).…”

Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by theDrosophila melanogaster suppressor of sableGene

“…In most intron-containing pre-mRNAs, the first intron is located near the beginning of the transcribed region (41), and an intron in this position appears to be important for expression at a high level. Studies in a variety of different systems indicate that cDNA transgenes are expressed at lower levels than their genomic counterparts in vivo and that expression of a cDNA can be elevated by inclusion of an intron near the 5Ј end of the gene (10,30,31,37,38,46). The magnitude of this effect varies, depending on sequences present in the RNA and the promoter that is used to direct transgene expression (33,35).…”

An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

“…In all species in which the I2 hairpin was present a pyrimidine-rich sequence was located between the BS and the 3 ′ SS. This polypyrimidine tract (PPT) has been shown to be important for splicing in higher eukaryotes (Roscigno et al 1993;Liu and Mertz 1996;Coolidge et al 1997). In contrast, in yeast species the 5 ′ SS, BS, and 3 ′ SS sequences are highly conserved, but the PPTs are not (Rymond and Rosbash 1985;Fouser and Friesen 1987).…”