Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells

Abstract

We have examined how splicing affects the expression of a range of human and nonhuman genes in vertebrate cells. Although our data demonstrate that splicing invariably enhances the level of gene expression, this positive effect is generally moderate. However, in the case of the human ␤-globin gene, splicing is essential for significant protein expression. In the absence of introns, 3 end processing is inefficient, and this appears to be causally linked to a significant decrease in the level of both nuclear and cytoplasmic 3 end-processed RNA. In contrast, splicing appears to only modestly enhance nuclear mRNA export. Consistent with this observation, intronless ␤-globin gene expression was only partially rescued by the insertion of retroviral nuclear mRNA export elements. Surprisingly, in the case of the highly intron dependent ␤-globin gene, the mRNA that did reach the cytoplasm was also only inefficiently translated if it derived from an intronless expression plasmid. Together, these data argue that splicing can increase gene expression by enhancing mRNA 3 end processing, and hence, mRNA production. Moreover, in the case of the highly intron-dependent ␤-globin gene, splicing also significantly enhanced the translational utilization of cytoplasmic ␤-globin mRNAs.

Frac-seq reveals isoform-specific recruitment to polyribosomes

“…One possibility is that alternative splicing alters the topology of these cis-acting regulatory elements, resulting in isoform-specific fates Staudacher et al 2011). Another possibility is that multifunctional cis-acting elements, such as the FN1 EDA splicing enhancer, may directly couple splicing decisions to translational control (Lu and Cullen 2003;Nott et al 2004;Barberan-Soler et al 2009). There is also new evidence supporting the idea of functional microRNA targets sites within coding exons (Hausser et al 2013).…”

Splicing promotes rapid and efficient mRNA export in mammalian cells

“…The specific mechanisms by which splicing promotes gene expression have also been extensively investigated, revealing that splicing affects several steps, including transcription, total mRNA levels, and 3Ј end processing (1,(16)(17)(18)(19)(20). In addition, splicing enhances both translation and localization of mRNA (13,14,21).Although splicing has also been reported to promote mRNA export, this functional connection has been controversial. In early studies, Ryu and Mertz (18) used transfection of mammalian cells followed by biochemical fractionation of the nucleus and cytoplasm to show that SV40 mRNAs generated by splicing were efficiently exported to the cytoplasm, whereas their cDNA counterparts were largely retained in the nucleus.…”

“…The specific mechanisms by which splicing promotes gene expression have also been extensively investigated, revealing that splicing affects several steps, including transcription, total mRNA levels, and 3Ј end processing (1,(16)(17)(18)(19)(20). In addition, splicing enhances both translation and localization of mRNA (13,14,21).…”

“…Similarly, splicing was required for efficient ␤-globin expression from SV40 expression vectors (4,5). Since then, numerous studies have reported a splicing-dependent enhancement of gene expression for multiple genes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Furthermore, this effect has been observed in Arabidopsis, Drosophila, maize cells, transgenic mice, and a variety of cell lines, indicating that it is a conserved and important feature of gene expression (7,9,11,13,15).…”

“…Since then, numerous studies have reported a splicing-dependent enhancement of gene expression for multiple genes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Furthermore, this effect has been observed in Arabidopsis, Drosophila, maize cells, transgenic mice, and a variety of cell lines, indicating that it is a conserved and important feature of gene expression (7,9,11,13,15). The specific mechanisms by which splicing promotes gene expression have also been extensively investigated, revealing that splicing affects several steps, including transcription, total mRNA levels, and 3Ј end processing (1,(16)(17)(18)(19)(20).…”