Regulation Of mRNA Export Research Articles

The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.

XPO1 Enables Adaptive Regulation of mRNA Export Required for Genotoxic Stress Tolerance in Cancer Cells.

XPO1 regulates the dynamic ribonucleoprotein nuclear export in response to genotoxic stress to support tolerance and can be targeted to enhance the sensitivity of cancer cells to endogenous and exogenous DNA damage. See related commentary by Knittel and Reinhardt, p. 3.

Abstract 6100: The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

Abstract Background: Fanconi Anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer susceptibility. FA gene mutations are also widespread somatically in non-FA cancer patients. While the FA pathway participates in DNA repair, it is also linked to R-loop metabolism and regulation. R-loops are co-transcriptional RNA:DNA hybrids that form as a result of replication-transcription collisions that are enhanced due to DNA damage, but their aberrant formation drives genomic instability and is an endogenous source of genotoxicity. However, the interplay between FA proteins, DNA repair, and R-loops remains mechanistically ill-defined. Disturbance of mRNA processing, export, and splicing has also been linked to R-loop-mediated genomic instability. The splicing factor SRSF1 participates in mRNA export through the Nuclear Export Factor 1 (NXF1). Aberrant splicing factors expression has been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), which clinically resembles FA. In the present study, we study the coupling of the DNA damage response to the prevention of pathogenic R-loops via mRNA export regulation through the FA pathway and SRSF1. Methods: Cells: FA-D2 mutant, corrected, and ubiquitin-dead mutant; HeLa. siRNA transfections to reduce SRSF1 and FANCD2 protein levels. Cell survival assays against DNA damaging agents. In vitro immunoprecipitation and ubiquitination assays using purified SRSF1 and ID2. DART assay, S9.6 immunofluorescence, and S9.6 slot-blot to measure R loops in cells. Immunoprecipitation of mRNPs used to detect mature mRNA-bound ribonucleoproteins. RNA FISH to measure mRNA export. Results: In this study, we provide strong evidence that the FA pathway coordinates the prevention of R-loop formation and subsequent genomic instability through its interaction with splicing factors via regulation of mRNA export. First, we demonstrate that knockdown of SRSF1, which is known to result in increased R-loops, also is associated with diminished activation of the FA pathway. Second, the central FA complex ID2 binds to SRSF1 in cells and in recombinant protein assays. Further, we demonstrate that SRSF1 activates the FA pathway by binding ID2 and stimulates its monoubiquitination in an RNA-dependent fashion. This activity is associated with co-localization in cells at transcriptional-DNA damage inducible R loops. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, cancer-associated SRSF1 mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export and R-loops accumulation. Conclusion: Our findings uncover a novel mechanism of FANCD2 and SRSF1 in the prevention of R-loop formation via coupling of mRNA export to the DNA damage response, thus preventing genomic instability. Citation Format: Anne Olazabal-Herrero, Fengshan Liang, Arijit Dutta, Yuxin Huang, Zhuobin Liang, Abhishek Gupta, Yaqun Teng, Li Lan, Xiaoyong Chen, Huadong Pei, Manoj Pillai, Patrick Sung, Gary Kupfer. The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6100.

The Xmas-2 Protein of Drosophila melanogaster Undergoes Cleavage into Two Fragments.

The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster, TREX-2 consists of four major proteins: Xmas-2, ENY2, PCID2, and Sem1p. The Xmas-2 protein is the core subunit of the complex, with which other TREX-2 subunits interact. Xmas-2 homologues were found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein, can undergo cleavage into two parts, probably during apoptosis. We showed that the Xmas-2 protein of D. melanogaster can also split into two fragments. The resulting fragments of the protein correspond to the two large Xmas-2 domains. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster.

THE XMAS-2 PROTEIN OF <i>DROSOPHILA MELANOGASTER</i> UNDERGOES CLEAVAGE INTO TWO FRAGMENTS

The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster TREX-2 consists of four main proteins: Xmas-2, ENY2, PCID2, and Sem1p. Xmas-2 protein is the core subunit of the complex with which other TREX-2 subunits interact. Xmas-2 homologues have been found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein can undergo cleavage into two parts, probably during apoptotic cell death. We showed that the Xmas-2 protein of D. melanogaster also can split into two fragments. The resulting fragments of the protein correspond to the two large domains of Xmas-2. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster.

Complex Formation between FANCD2 and the Splicing Factor SRSF1 Helps Prevent R-Loop Accumulation through mRNA Export Regulation

Complex Formation between FANCD2 and the Splicing Factor SRSF1 Helps Prevent R-Loop Accumulation through mRNA Export Regulation

Emerging molecular functions and novel roles for the DEAD-box protein Dbp5/DDX19 in gene expression.

The DEAD-box protein (DBP) Dbp5, a member of the superfamily II (SFII) helicases, has multiple reported roles in gene expression. First identified as an essential regulator of mRNA export in Saccharomyces cerevisiae, the enzyme now has reported functions in non-coding RNA export, translation, transcription, and DNA metabolism. Localization of the protein to various cellular compartments (nucleoplasm, nuclear envelope, and cytoplasm) highlights the ability of Dbp5 to modulate different stages of the RNA lifecycle. While Dbp5 has been well studied for > 20years, several critical questions remain regarding the mechanistic principles that govern Dbp5 localization, substrate selection, and functions in gene expression. This review aims to take a holistic view of the proposed functions of Dbp5 and evaluate models that accommodate current published data.

METTL1-mediated m7G methylation maintains pluripotency in human stem cells and limits mesoderm differentiation and vascular development

Background7-Methylguanosine (m7G) is one of the most conserved modifications in nucleosides within tRNAs and rRNAs. It plays essential roles in the regulation of mRNA export, splicing, and translation. Recent studies highlighted the importance of METTL1-mediated m7G tRNA methylome in the self-renewal of mouse embryonic stem cells (mESCs) through its ability to regulate mRNA translation. However, the exact mechanisms by which METTL1 regulates pluripotency and differentiation in human induced pluripotent stem cells (hiPSCs) remain unknown. In this study, we evaluated the functions and underlying molecular mechanisms of METTL1 in regulating hiPSC self-renewal and differentiation in vivo and in vitro.MethodsBy establishing METTL1 knockdown (KD) hiPSCs, gene expression profiling was performed by RNA sequencing followed by pathway analyses. Anti-m7G northwestern assay was used to identify m7G modifications in tRNAs and mRNAs. Polysome profiling was used to assess the translation efficiency of the major pluripotent transcription factors. Moreover, the in vitro and in vivo differentiation capacities of METTL1-KD hiPSCs were assessed in embryoid body (EB) formation and teratoma formation assays.ResultsMETTL1 silencing resulted in alterations in the global m7G profile in hiPSCs and reduced the translational efficiency of stem cell marker genes. METTL1-KD hiPSCs exhibited reduced pluripotency with slower cell cycling. Moreover, METTL1 silencing accelerates hiPSC differentiation into EBs and promotes the expression of mesoderm-related genes. Similarly, METTL1 knockdown enhances teratoma formation and mesoderm differentiation in vivo by promoting cell proliferation and angiogenesis in nude mice.ConclusionOur findings provided novel insight into the critical role of METTL1-mediated m7G modification in the regulation of hiPSC pluripotency and differentiation, as well as its potential roles in vascular development and the treatment of vascular diseases.

Regulation of mRNA export through API5 and nuclear FGF2 interaction.

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5–FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.

Structural and functional analysis of mRNA export regulation by the nuclear pore complex

The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP6, unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.

Genome-wide mapping of infection-induced SINE RNAs reveals a role in selective mRNA export.

Short interspersed nuclear elements (SINEs) are retrotransposons evolutionarily derived from endogenous RNA Polymerase III RNAs. Though SINE elements have undergone exaptation into gene regulatory elements, how transcribed SINE RNA impacts transcriptional and post-transcriptional regulation is largely unknown. This is partly due to a lack of information regarding which of the loci have transcriptional potential. Here, we present an approach (short interspersed nuclear element sequencing, SINE-seq), which selectively profiles RNA Polymerase III-derived SINE RNA, thereby identifying transcriptionally active SINE loci. Applying SINE-seq to monitor murine B2 SINE expression during a gammaherpesvirus infection revealed transcription from 28 270 SINE loci, with ∼50% of active SINE elements residing within annotated RNA Polymerase II loci. Furthermore, B2 RNA can form intermolecular RNA–RNA interactions with complementary mRNAs, leading to nuclear retention of the targeted mRNA via a mechanism involving p54nrb. These findings illuminate a pathway for the selective regulation of mRNA export during stress via retrotransposon activation.

Temporal and spatial regulation of mRNA export: Single particle RNA‐imaging provides new tools and insights

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression.

Genome-wide DNA methylation in neonates exposed to maternal depression, anxiety, or SSRI medication during pregnancy

Despite the high prevalence of depression, anxiety, and use of antidepressant medications during pregnancy, there is much uncertainty around the impact of high levels of distress or antidepressant medications on the developing fetus. These intrauterine exposures may lead to epigenetic alterations to the DNA during this vulnerable time of fetal development, which may have important lifetime health consequences. In this study we investigated patterns of genome-wide DNA methylation using the Illumina Infinium Human Methylation450 BeadChip in the umbilical cord blood of neonates exposed to non-medicated maternal depression or anxiety (n = 13), or selective serotonin reuptake inhibitors (SSRIs) during pregnancy (n = 22), relative to unexposed neonates (n = 23). We identified 42 CpG sites with significantly different DNA methylation levels in neonates exposed to non-medicated depression or anxiety relative to controls. CpG site methylation was not significantly different in neonates exposed to SSRIs relative to the controls, after adjusting for multiple comparisons. In neonates exposed either to non-medicated maternal depression or SSRIs, the vast majority of CpG sites displayed lower DNA methylation relative to the controls, but differences were very small. A gene ontology analysis suggests significant clustering of the top genes associated with non-medicated maternal depression/anxiety, related to regulation of transcription, translation, and cell division processes (e.g., negative regulation of translation in response to oxidative stress, regulation of mRNA export from the nucleus, regulation of stem cell division). While the functional consequences of these findings are yet to be determined, these small DNA methylation differences may suggest a possible role for epigenetic processes in the development of neonates exposed to non-medicated maternal depression/anxiety.

The nuclear mRNA export receptor Mex67‐Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.

Regulation of mRNA export by the PI3 kinase/AKT signaling pathway

mRNA export from the nucleus is critical for gene expression, but its regulation is not well understood. We studied the regulation of mRNA export by the PI3Kinase/AKT pathway, a controller of proliferation and survival often deregulated in cancer. UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the TRanscription/EXport (TREX) complex at the 5′ end of nuclear mRNA. NXF1 subsequently binds at nuclear pores for mRNP export. We used Fluorescence Recovery after Photobleaching (FRAP) to measure binding of these export factors in complexes at an integrated array of actively transcribed β‐globin genes and at RNA splicing speckled domains. Inhibition of the PI3 kinase/AKT pathway decreased the fraction of UAP56 and ALY/REF in complexes at β‐globin transcription sites. Pathway inhibition did not significantly affect localization of export factors at the β‐globin genes, but did affect localization of the splicing factor SRm300. qRT‐PCR results showed pathway inhibition did not affect turnover rates for selected mRNAs but may affect exon inclusion for β‐globin. These data clarify the regulation of mRNA metabolism by PI3 kinase/AKT signaling. This work was supported by NCI (PO1CA82834).

Signal transduction and signaling networks

Cellular signaling can be complex and dynamic. In the past several decades, it has become clear that many signal transduction pathways are not simple one-way transmissions of upstream stimuli, but are mediated through extremely complicated networks. Our next challenge is to move beyond the static biochemical description of networks into developing a functional quantitative understanding of their behavior. In this Minisymposium, we presented diverse approaches for beginning to achieve this goal. Galit Lahav (Harvard Medical School) opened the session and demonstrated how the temporal dynamics of the tumor suppressor protein p53 in single cells affect cell fate decisions. Cells that naturally show a series of p53 pulses in response to irradiation and recover from the damage were perturbed to instead show sustained p53. This perturbation led to activation of different target genes and pushed cells toward permanent cell cycle arrest. p53 dynamics therefore contribute to transferring information in cells and provide a new axis for pushing cells toward a specific cellular outcome. Andrei V. Karginov (University of Illinois at Chicago) focused on the spatiotemporal regulation of the Src signaling pathway by engineering a chimera of the Src kinase domain and an insertable FKBP12 protein, iFKBP. By fusing the FKBP12-rapamycin binding domain (FRB) to the specific downstream effectors, he restricted the Src activation to the complex it forms with FRB-bearing downstream targets. He presented evidence that a specific complex of Src and FAK leads to focal adhesion, while a complex of Src and p130Cas leads to filopodia formation. Robin E. Lee (Dana-Farber Cancer Institute) demonstrated that, upon tumor necrosis factor-α (TNF-α) stimulation, the early-response genes of NF-κB use memory to assess NF-κB activation. He analyzed the nuclear translocation dynamics of p65, a major NF-κB transcription factor, and then the number of mRNA transcripts for target genes in the same cells. The gene transcriptions of three early-response genes, IL8, A20, and IκB α, were regulated by the fold changes of nuclear NF-κB. His results, supported by computational modeling, suggest that competitive transcription factor–DNA interactions can provide memory of pre-ligand NF-κB states, allowing fold-change signal detection. The NF-κB signaling pathway has been also shown to involve ubiquitin modification of important molecules. Fumiyo Ikeda (Institute of Molecular Biotechnology) reported that an atypical type of ubiquitin chain, linearly linked chains, plays a critical role in the regulation of a Sharpin-dependent, anti-apoptotic pathway downstream of TNF. She presented a critical apoptosis signaling molecule, FADD, as a novel substrate of the Sharpin-containing E3 ligase that plays a role in the regulation of the apoptotic cascade. Jeffery A. Nickerson (University of Massachusetts Medical School) focused on the regulation of mRNA export by phosphatidylinositol 3-kinase/protein kinase B (PI3 kinase/Akt) signal transduction. His group implemented a fluorescence recovery after photobleaching system for screening signal transduction pathways that regulate mRNA nuclear export and the binding of the exon junction complex core proteins. By combining the analysis of mRNA export to the cytoplasm, they showed that the PI3 kinase/AKT pathway regulates not only mRNA export complex formation, but also the rate of mRNA nuclear export. Anna Podgornaia (Massachusetts Institute of Technology) tackled a challenging question to understand the transient protein–protein interaction surface that determines protein pairing by using a systematic mutagenesis approach. She focused on the histidine kinase PhoQ and its cognate response regulator PhoP, which together regulate bacterial signaling. Out of 160,000 PhoQ variants completely randomized at four residues, 4600 variants were signal-responsive. She is exploring why all these 4600 sequences are not used by extant PhoQ orthologues, which only have 260 different interface sequences.

Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway

UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5' end of a nuclear mRNA but are also reported to associate with the exon junction complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly, we used fluorescence recovery after photobleaching to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the phosphatidylinositide 3-kinase (PI3 kinase)/AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intronless histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the endoplasmic reticulum or to mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and promote the nuclear retention of some mRNAs.

A new regulatory pathway of mRNA export by an F‐box protein, Mdm30

Regulatory role of ubiquitylation has been implicated in various steps of gene expression and mRNA export is not an exception. Specificity of this modification is conferred by E3 ligases whose function is dictated by specific domains they possess. Role of HECT domain containing E3 ligases like Tom1 and Rsp5 have previously been implicated in regulation of mRNA export. However, pertaining to diversity of E3 ligases, the roles of other ligases in this aspect of gene expression is unknown. Interestingly, our recent work established the role of an F‐box protein, Mdm30, in stimulation of mRNA export. F‐box proteins are bonafide members of RING domain containing SCF (Skp1‐Cullin‐F box) ligase complex. To elucidate the mechanism‐of‐action of Mdm30, we carried out a series of experiments. We find that Mdm30 ubiquitylates the Sub2 component of Transcription/Export complex (TREX) in SCF dependent manner. Ubiquitylated‐Sub2 is subsequently degraded by 26S proteasome complex. Such targeted degradation of Sub2 enhances the recruitment of mRNA export adaptor, Yra1, onto active gene to promote mRNA export. Collectively, these results provide a new regulatory pathway of mRNA export, and will be presented in this conference.Funding: Excellence in Academic Medicine (EAM) from the School of Medicine, Southern Illinois University.

Evolutionary development of redundant nuclear localization signals in the mRNA export factor NXF1

In human cells, the mRNA export factor NXF1 resides in the nucleoplasm and at nuclear pore complexes. Karyopherin β2 or transportin recognizes a proline-tyrosine nuclear localization signal (PY-NLS) in the N-terminal tail of NXF1 and imports it into the nucleus. Here biochemical and cellular studies to understand the energetic organization of the NXF1 PY-NLS reveal unexpected redundancy in the nuclear import pathways used by NXF1. Human NXF1 can be imported via importin β, karyopherin β2, importin 4, importin 11, and importin α. Two NLS epitopes within the N-terminal tail, an N-terminal basic segment and a C-terminal R-X(2-5)-P-Y motif, provide the majority of binding energy for all five karyopherins. Mutation of both NLS epitopes abolishes binding to the karyopherins, mislocalized NXF1 to the cytoplasm, and significantly compromised its mRNA export function. The understanding of how different karyopherins recognize human NXF1, the examination of NXF1 sequences from divergent eukaryotes, and the interactions of NXF1 homologues with various karyopherins reveals the evolutionary development of redundant NLSs in NXF1 of higher eukaryotes. Redundancy of nuclear import pathways for NXF1 increases progressively from fungi to nematodes and insects to chordates, potentially paralleling the increasing complexity in mRNA export regulation and the evolution of new nuclear functions for NXF1.

Zinc finger‐containing glycine‐rich RNA‐binding protein in Oryza sativa has an RNA chaperone activity under cold stress conditions

The rice (Oryza sativa) genome harbours three genes encoding CysCysHisCys (CCHC)-type zinc finger-containing glycine-rich RNA-binding proteins, designated OsRZ proteins, but their importance and physiological functions remain largely unknown. Here, the stress-responsive expression patterns of OsRZs were assessed, and the biological and cellular functions of OsRZs were evaluated under low temperature conditions. The expression levels of the three OsRZs were up-regulated by cold stress, whereas drought or high salt stress did not significantly alter its transcript level. OsRZ2 complemented the cold sensitivity of BX04 Escherichia coli cells under low temperatures, and had DNA-melting activity and transcription anti-termination activity, thereby indicating that OsRZ2 possesses an RNA chaperone activity. By contrast, neither OsRZ1 nor OsRZ3 harboured these activities. Ectopic expression of OsRZ2, but not OsRZ3, in cold-sensitive Arabidopsis grp7 knockout plants rescued the grp7 plants from cold and freezing damage, and OsRZ2 complemented the defect in mRNA export from the nucleus to the cytoplasm in grp7 mutant during cold stress. The present findings support the emerging idea that the regulation of mRNA export is one of the adaptive processes in plants under stress conditions, and RNA chaperone functions as a regulator in mRNA export under cold stress conditions.