Splicing promotes rapid and efficient mRNA export in mammalian cells

Valencia, Patricia; Dias, Anusha P.; Reed, Robin

doi:10.1073/pnas.0800250105

Cited by 210 publications

(224 citation statements)

References 41 publications

(74 reference statements)

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“…The HIV-1 proteins env, vpu, vif, and vpr are translated from singly spliced HIV-1 mRNA; Tat, nef, and rev derive from doubly spliced mRNA; and the proteins gag and gag-pol are translated from unspliced mRNA (Meyer and Malim 1994;Cullen 1998). On one hand, splicing is an essential process that also stimulates nucleocytoplasmic transport (Katahira et al 1999;Valencia et al 2008). On the other hand, pre-mRNAs that have initiated spliceosomal assembly are retained in the nucleus until splicing is completed (Galy et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Nawroth¹,

Mueller²,

Basyuk³

et al. 2013

RNA

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The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post-or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.

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Section: Introductionmentioning

confidence: 99%

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Nawroth¹,

Mueller²,

Basyuk³

et al. 2013

RNA

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“…Other studies have implicated a role for mRNA splicing in mediating increased nuclear mRNA export. 16,23,41,42 However, our experiments in cultured cells failed to reveal an effect of splicing on the nuclear-cytoplasmic distribution of mRNA. Finally, there is increasing evidence that mRNA splicing enhances mRNA translational yield (ie, the number of protein molecules per mRNA molecule).…”

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confidence: 40%

Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium

Yuan

Janes

Beeler

et al. 2013

Blood

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Key Points• RNA splicing of the first intron of the von Willebrand factor gene is essential for expression in the endothelium.• RNA splicing may play a role in mediating endothelial cell heterogeneity.We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human b-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium. (Blood. 2013;121(21):4404-4412) Introduction Von Willebrand factor (vWF) is a plasma protein that mediates platelet hemostatic function and stabilizes coagulation factor VIII. vWF plays a particularly important role in platelet adhesion and aggregation at sites of high shear rates.1 Expression of vWF is restricted to endothelial cells and platelets. vWF is differentially expressed in the vasculature. For example, vWF protein and mRNA levels are higher in veins, compared with arteries, 2,3 and in venules compared with arterioles. 4 Within the microvasculature, vWF is expressed at particularly low levels in the liver. Expression of vWF also varies between neighboring endothelial cells. 5 The elucidation of the mechanisms underlying vWF expression may provide important insights into the molecular basis of vascular diversity.The structural organization of the mouse, human, and bovine vWF promoter-proximal region is closely related. [6][7][8] The first exon (11 to 1 246 in human vWF) encodes the 59 untranslated sequence. The second exon contains the ATG translational start site. Exons 1 and 2 are separated by an intron (1247 to 11475 in human vWF). To study mechanisms of vWF gene regulation, we have previously used a plug-insocket approach in which a single copy of a vWF promoter-LacZ cassette is targeted to the Hprt locus of mice. Using this strategy, we showed that a region of the human vWF gene (termed vWF2, between 22182 and the end of the first intron) directed expression in the vasculature of the brain, heart and skeletal muscle, but not in other organs such as aorta, lung, liver, spleen, ...

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“…Silencing experiments were performed in HeLa cells stably transfected with the ß-globin gene lacking natural introns and therefore expressing cDNA transcripts that do not undergo splicing (Valencia et al, 2008). The assay was validated by depleting NXF1.…”

Section: Molecular Biology Of the Cell 5218mentioning

confidence: 99%

“…The cytoplasmic (CЈ) amount of mRNA was calculated as follows: CЈ ϭ 1 Ϫ [(An Sn)/(Ac Sc)]. C/N ratios were calculated as C/N ϭ CЈ/(1 Ϫ CЈ) (for reference see Valencia et al, 2008).…”

Section: Fluorescent In Situ Hybridizationmentioning

confidence: 99%

“…The cytoplasmic (CЈ) amount of mRNA was calculated as follows: CЈ ϭ 1 Ϫ [(An Sn)/(Ac Sc)]. C/N ratios were calculated as C/N ϭ CЈ/(1 Ϫ CЈ) (for reference see Valencia et al, 2008).For the visualization of poly(A)ϩ RNA, HeLa cells were grown in six-well plates on coverslips and transfected with the different plasmids, as indicated. Seven days past transfection, the cells were washed two times in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ) before they were fixed for 10 min at RT in 3.7% paraformaldehyde (PFA).…”

mentioning

confidence: 99%

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Mammalian pre-mRNA 3′ End Processing Factor CF I_m68 Functions in mRNA Export

Ruepp

Aringhieri

Vivarelli

et al. 2009

MBoC

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Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3 end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3 end processing complex, CF I m 68, stimulates mRNA export. CF I m 68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF I m 68 may act as a novel adaptor for NXF1/TAP, we show that CF I m 68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF I m 68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3 end processing factor CF I m 68 in mRNA export. INTRODUCTIONThe removal of introns by splicing as well as cleavage and polyadenylation at the 3Ј end of RNA polymerase II primary transcripts (pre-mRNAs) are usually required before they can be exported from the nucleus as mature mRNAs (Erkmann and Kutay, 2004). This observation has suggested that transport factors interact with the RNA during premRNA processing. Indeed, recent discoveries have lent support to this hypothesis. The splicing reaction deposits on the mRNA a specific subset of proteins called the exon junction complex (EJC, for review see Tange et al., 2004). REF, a component of the EJC, facilitates mRNA export by interacting with the mRNA export factor NXF1 (also called TAP, for review, see Reed and Hurt, 2002). NXF1 was originally identified as the export receptor for type D retroviral RNAs that associate with NXF1 through a sequence-specific interaction with the constitutive transport element (CTE). However, NXF1 recruitment on cellular mRNAs requires adaptor proteins such as Aly/REF (hereafter named REF). In yeast Mex67 (the homolog of NXF1) is recruited by Yra1 (homolog of REF), which is also essential for the export of poly(A) RNA in Saccharomyces cerevisiae. In contrast in metazoans, REF is dispensable for bulk mRNA export. This raises the possibility that multiple and partially redundant adaptor proteins may be responsible for the recruitment of NXF1. Indeed, spliceosomal proteins, including U2AF35 (Zolotukhin et al., 2002) and some members of the SR family of splicing factors, were shown to interact with NXF1 and act as adaptors for NXF1-dependent export of poly(A) mRNAs (Huang and Steitz, 2001;Huang et al., 2003;Lai and Tarn, 2004;Hargous et al., 2006;Tintaru et al., 2007).Several observations have linked 3Ј end cleavage and polyadenylation to mRNA export (for review see Zhao et al., 1999). For example, RNA polymerase II reporter transcripts lacking a polyadenylation signal are retained in the nucleus of yeast cells. Positioning a...

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Splicing promotes rapid and efficient mRNA export in mammalian cells

Cited by 210 publications

References 41 publications

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium

Mammalian pre-mRNA 3′ End Processing Factor CF I_m68 Functions in mRNA Export

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Splicing promotes rapid and efficient mRNA export in mammalian cells

Cited by 210 publications

References 41 publications

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium

Mammalian pre-mRNA 3′ End Processing Factor CF Im68 Functions in mRNA Export

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Mammalian pre-mRNA 3′ End Processing Factor CF I_m68 Functions in mRNA Export