Trafficking of mRNAs containing ALREX-promoting elements through nuclear speckles

Abstract

In vertebrates, the majority of mRNAs that encode secreted, membrane-bound or mitochondrial proteins contain RNA elements that activate an alternative mRNA nuclear export (ALREX) pathway. Here we demonstrate that mRNAs containing ALREX-promoting elements are trafficked through nuclear speckles. Although ALREX-promoting elements enhance nuclear speckle localization, additional features within the mRNA largely drive this process. Depletion of two TREX-associated RNA helicases, UAP56 and its paralog URH49, or inhibition of the TREX-associated nuclear transport factor, TAP, not only inhibits ALREX, but also appears to trap these mRNAs in nuclear speckles. mRNAs that contain ALREX-promoting elements associate with UAP56 in vivo. Finally, we demonstrate that mRNAs lacking a poly(A)-tail are not efficiently exported by the ALREX pathway and show enhanced association with nuclear speckles. Our data suggest that within the speckle, ALREX-promoting elements, in conjunction with the poly(A)-tail, likely stimulate UAP56/URH49 and TAP dependent steps that lead to the eventual egress of the export-competent mRNP from these structures.

Splicing promotes the nuclear export of β-globin mRNA by overcoming nuclear retention elements

“…These observations indicated that either the ftz RNA has an additional nuclear export-promoting element, or that when nuclear retention elements are eliminated, all mRNAs become substrates for nuclear export. These two possibilities were supported by the finding that UAP56, a central component of the TREX complex, is efficiently loaded onto ftz without the requirement of splicing (Taniguchi and Ohno 2008;Akef et al 2013;Lee et al 2015).…”

“…In contrast, βG-Δi was mainly nuclear, and the fusion of full-length MHC-ftz-Δi to βG-Δi was primarily found in the cytoplasm (Fig. 1B,C), as we had previously seen (Akef et al 2013).…”

“…Surprisingly, we found mRNA produced from a gene containing an SSCR derived from the mouse MHC gene h2kb fused to βG-Δi (MHC-βG-Δi) was not efficiently exported (Akef et al 2013). In contrast, mRNA produced from a fusion gene containing this SSCR, the ftz ORF (thus lacking its nuclear retention element), and βG-Δi (MHCftz-Δi-βG-Δi) was efficiently exported (Akef et al 2013). In light of these results, we reasoned that ftz might contain a cis-element that promotes nuclear export.…”

“…This would explain the relative degrees of cytoplasmic accumulation where MHC-ftz-βG-Δi is more cytoplasmic than any of the fusion constructs (F1-βG-Δi through F4-βG-Δi), which in turn are more cytoplasmic than MHC-βG-Δi (Akef et al 2013), which is slightly more cytoplasmic than βG-Δi. Indeed, it had been previously found that certain RNA-binding proteins are differentially recruited to RNAs of different figure. (B-E) Plasmids containing the indicated constructs were transfected into human U2OS cells.…”

“…As we have done previously (Akef et al 2013;Lee et al 2015), we expressed various reporters in U2OS cells and then from these lysates immunoprecipitated UAP56 (see Fig. 7A), a central component of the TREX complex, and analyzed the precipitated RNA by RT-qPCR.…”