Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation.

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3' splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.

Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5' proximal-intron-minus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, N1-methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ∼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N1-methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC.

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

Most current models of mRNA nuclear export in vertebrate cells assume that an mRNA must have specialized signals in order to be exported from the nucleus. Under such a scenario, mRNAs that lack these specialized signals would be shunted into a default pathway where they are retained in the nucleus and eventually degraded. These ideas were based on the selective use of model mRNA reporters. For example, it has been shown that splicing promotes the nuclear export of certain model mRNAs, such as human ß-globin, and that in the absence of splicing, the cDNA-derived mRNA is retained in the nucleus and degraded. Here we provide evidence that ß-globin mRNA contains an element that actively retains it in the nucleus and degrades it. Interestingly, this nuclear retention activity can be overcome by increasing the length of the mRNA or by splicing. Our results suggest that contrary to many current models, the default pathway for most intronless RNAs is to be exported from the nucleus, unless the RNA contains elements that actively promote its nuclear retention.

RanBP2/Nup358 potentiates the translation of a subset of mRNAs encoding secretory proteins.

In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX) pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER) and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins.

Genome analysis reveals interplay between 5'UTR introns and nuclear mRNA export for secretory and mitochondrial genes.

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5' end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR-containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX-promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5' untranslated region (5'UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export.

Nuclear export as a key arbiter of "mRNA identity" in eukaryotes.

Over the past decade, various studies have indicated that most of the eukaryotic genome is transcribed at some level. The pervasiveness of transcription might seem surprising when one considers that only a quarter of the human genome comprises genes (including exons and introns) and less than 2% codes for protein. This conundrum is partially explained by the unique evolutionary pressures that are imposed on species with small population sizes, such as eukaryotes. These conditions promote the expansion of introns and non-functional intergenic DNA, and the accumulation of cryptic transcriptional start sites. As a result, the eukaryotic gene expression machinery must effectively evaluate whether or not a transcript has all the hallmarks of a protein-coding mRNA. If a transcript contains these features, then positive feedback loops are activated to further stimulate its transcription, processing, nuclear export and ultimately, translation. However if a transcript lacks features associated with "mRNA identity", then the RNA is degraded and/or used to inhibit further transcription and translation of the gene. Here we discuss how mRNA identity is assessed by the nuclear export machinery in order to extract meaningful information from the eukaryotic genome. In the process, we provide an explanation of why certain sequences that are enriched in protein-coding genes, such as the signal sequence coding region, promote mRNA nuclear export in vertebrates. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

How T cells go rogue in the absence of Roquins.

Roquin-1 and Roquin-2 are RNA-binding proteins essential for modulating T cell activity. Indeed, Roquin dysfunction has been linked to autoimmunity in mice. Essig and colleagues (2017) determine their functions in Foxp3+ T regulatory cells and uncover novel mechanisms of Roquin-mediated regulation of its target mRNAs (1).

A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies.

Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer's vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function.

The consensus 5' splice site motif inhibits mRNA nuclear export.

In eukaryotes, mRNAs are synthesized in the nucleus and then exported to the cytoplasm where they are translated into proteins. We have mapped an element, which when present in the 3'terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5'splice site motif that is used to define the start of introns. Previously it was shown that when this motif is retained in the mRNA, it causes defects in 3'cleavage and polyadenylation and promotes mRNA decay. Our new data indicates that this motif also inhibits nuclear export and promotes the targeting of transcripts to nuclear speckles, foci within the nucleus which have been linked to splicing. The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export. Genome wide analysis of human mRNAs, lncRNA and eRNAs indicates that this motif is depleted from naturally intronless mRNAs and eRNAs, but less so in lncRNAs. This motif is also depleted from the beginning and ends of the 3'terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5'splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise.

Trafficking of mRNAs containing ALREX-promoting elements through nuclear speckles.

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.