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

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

A Common Class of Transcripts with 5\u27-Intron Depletion, Distinct Early Coding Sequence Features, and N1-Methyladenosine Modification [preprint]

Introns are found in 5\u27 untranslated regions (5\u27UTRs) for 35% of all human transcripts. These 5\u27UTR introns are not randomly distributed: genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5\u27UTR 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\u27UTR intron status, we developed a classifier that can predict 5\u27UTR intron status with \u3e80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5\u27 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\u27 cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the Exon Junction Complex (EJC) at non-canonical 5\u27 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\u27 proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N1-methyladenosines in the early coding region, and enrichment for non-canonical binding by the Exon Junction Complex

A common class of transcripts with 5\u27-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification

Introns are found in 5\u27 untranslated regions (5\u27UTRs) for 35% of all human transcripts. These 5\u27UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5\u27UTR 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\u27UTR intron status, we developed a classifier that can predict 5\u27UTR intron status with \u3e 80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5\u27 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\u27 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\u27 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 approximately 20% of human transcripts. This class is defined by depletion of 5\u27 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

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

Investigating mRNA Sequence Features that Regulate Nuclear Export

In eukaryotes, the nucleus divides the cell into two compartments: the nucleoplasm, where RNA is synthesized, processed and packaged, and the cytoplasm, where mature mRNA is translated into proteins. The mechanisms that determine whether an mRNA should be exported from the nucleus to the cytoplasm are poorly understood. The best established model postulated that mRNAs need to be spliced or to contain nuclear export-promoting sequences so as to be efficiently transported to the cytoplasm. However, this model suffered several problems since it was known that many intronless mRNAs are efficiently exported to the cytoplasm independently of splicing and they do not contain any known nuclear export-promoting sequences. In this thesis, I sought to dissect the sequence features within a transcript that determine its cellular localization. I discovered that intronless β-Globin (βG) mRNA contains a nuclear retention element that inhibits its transport to the cytoplasm. This nuclear retention element can be overcome when βG mRNA is either spliced or its length extended. The mRNA nuclear export factor UAP56 binds to several intronless mRNAs including βG mRNA independently of splicing. I also discovered that UAP56 is required for the egress of intronless mRNAs from nuclear speckles. My results suggest that most mRNAs are exported to the cytoplasm independently of splicing unless they contain a nuclear retention element.Ph.D

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

The 5’SS motif decreases mRNA stability and promotes nuclear retention.

<p>(A) U2OS cells were transfected with various <i>ftz</i> and <i>β-globin</i> constructs. 18–24 hrs post transfection, RNA was isolated, separated by agarose gel electrophoresis and probed with radiolabelled probes against α<i>-tubulin</i>, <i>ftz</i> and <i>β-globin</i> mRNAs. Note that the mRNAs containing the <i>V5-His</i> element were slightly larger, likely due to the presence of this extra sequence. (B-C) Transfected cells were treated with α-amanitin, at a concentration that completely inhibits transcription (see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122743#pone.0122743.ref030" target="_blank">30</a>]) for various amounts of time in order to determine the half-life of the mRNA. Levels of <i>ftz</i> mRNA were monitored by northern blot (B) or FISH (C). Each bar in C represents the average and standard error of at least 60 cells. (D-E) Plasmids containing <i>c-ftz-Δi</i> with and without the <i>V5-His</i> element were microinjected into the nuclei of U2OS cells. After allowing mRNA synthesis for 20 min, cells were treated with α-amanitin and mRNA levels were monitored over time by FISH. (D) To determine whether the <i>V5-His</i> sequence promotes the degradation of a subset of newly synthesized mRNAs the ratio of <i>c-ftz-Δi</i> and <i>c-ftz-Δi-V5-His</i> were plotted over time. Each point is the average and standard error of five independent experiments, each of which consist of 15–30 cells. Note that the relative level of <i>c-ftz-Δi-V5-His</i> decreases over the first 60 min until 40–50% of the mRNA remains, after which point the ratio is stable. (E) To determine whether the <i>V5-His</i> element inhibits nuclear export the percentage of cytoplasmic mRNA was plotted over time. Again, each point is the average and standard error of five independent experiments, each of which consist of 15–30 cells. Note that at the 1 hr time point, at which point most 5’SS-promoted decay has already occurred, the large fraction of nuclear mRNA (representing 70% of the total mRNA) is not efficiently exported. (F-G) Experiments were performed as in (D-E) except the microinjected plasmids contained <i>βG-i</i>, with and without the <i>V5-His</i> element, and α-amanitin was added 30 min post-injection. The ratio of total <i>βG-i-V5-His</i> to <i>βG-I</i> FISH signal (F) and the percentage of cytoplasmic mRNA (G) was plotted over time. (H-I) <i>MHC-ftz-Δi</i> mRNA, lacking the <i>V5-His</i> element (“-”), or containing either the original or mutant version of the <i>V5-His</i> element, were synthesized, capped and polyadenylated <i>in vitro</i>. The mRNAs were them microinjected into nuclei and the mRNA export (F) and total fluorescence (G) was monitored over time by FISH. Each point is the average and standard error of three independent experiments, each of which consist of 15–30 cells.</p

Characterizing the nuclear retained mRNP state.

<p>(A) Plasmids containing the indicated genes were microinjected into nuclei. After 2 hrs, cells were fixed and stained for <i>β-globin</i> mRNA by FISH and SC35 by immunofluorescence. Each row represents a single field of view. The color overlay shows mRNA in red and SC35 in green. Speckles containing mRNA are indicated by0020arrows (Scale bar = 10μm). (B) Microinjected cells were analyzed for the distribution of mRNAs into nuclear speckles. Individual nuclear speckles, as determined by SC35 staining were analyzed for mRNA content by Pearson Correlation Analysis. For details on the analysis please see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122743#sec010" target="_blank">methods</a> section. Each bar is the average and standard error of the mean of three experiments, each of which consist of 150–200 speckles analyzed from 15–20 cells. Note that nuclear-speckle association is enhanced by the <i>V5-His</i> element even in the presence of splicing. (C) The amount of various mRNA present in nuclear speckles (as defined by the brightest 10% pixels in the nucleus, using SC35 immunofluorescence—see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122743#sec010" target="_blank">methods</a> section and Akef et al., 2013 for details) as a percentage of either the total nuclear (“spec/nuc”) or total cellular (“spec/tot”) mRNA level in cells 1 hr post-microinjection. Each data point represents the average and standard error of the mean of 10–20 cells. Note that <i>β-globin-Δi</i> was not enriched in speckles as ~10% of the nuclear RNA fluorescence was present in these regions, which represents 10% of the total nuclear area. (D-E) Similar to (A-B), except that the co-localisation of <i>c-ftz-Δi</i> +/- <i>V5-His</i> RNA with nuclear speckles was analysed. As a control, the colocalization of SC35-positive speckles with microinjected 70kD dextran conjugated to oregon green dye (“OG”) was analyzed.</p