| Research BackgroundCorrect RNA localization is vital to its function.In human genome,95%of protein coding genes contain multi-exons,the other 5%are genes with single exon.All mRNAs encoded by multi-exon or single exon genes need to be exported from the nucleus to the cytoplasm for translation.It is now widely accepted that TREX-TAP pathway is fundamental for the export of transcripts from multi-exon genes,with TREX complex recruited during splicing.More specifically,cap-binding complex(CBC)binds the cap structure after transcription,CBC component CBP80 interacts with TREX component ALYREF during splicing to recruit TREX complex to the 5' of spliced mRNA,later,ALYREF interacts with TAP/p15(NXF1:NXT1)dimer at nuclear pore and leads to the export of spliced mRNA from 5' to 3'.In addition to ALYREF,SR(Ser/Arg-rich)proteins such as 9G8/SRSF7 and SRp20/SRSF3 have been reported to serve as adaptor in mRNA export which facilitate the recruitment of general export factor.In contrast to deep insights in mechanisms on the export of spliced mRNAs,much less is known for the export of naturally intronless mRNAs in human.How these mRNAs are exported efficiently at the absence of splicing has puzzled the field for decades.Previous studies using four naturally intronless reporters from human support a model of sequence dependent recruitment of export machinery,in which cytoplasmic accumulation regions(CAR)critical for the export were identified in the coding region of the transcripts.The CAR or the consensus CAR-E facilitates the export of naturally intronless mRNAs via TREX-TAP pathway.Other previous work to explore mechanism for unspliced RNA export focused on viral RNAs and pinpointed to the importance of RNA sequences/motifs.So far motifs mapped include post-transcriptional regulatory element(PRE),constitutive transport element(CTE),,the direct repeat(DR),pre-mRNA processing enhancer(PPE).These motifs have been reported to interact mostly with mRNA export receptor TAP/NXF1 or in selective cases CRM1 often at the presence of additional adaptors.For instance,PRE utilizes ICP27 adaptor to bind TAP/NXF1 and binding of hnRNP-L to PPE enhances export.Likewise,CTE-RNA recognition is facilitated by NXF1:NXT1 dimerization and ZC3H18 recruited to the sub-element of PRE is essential for association of TREX and RNA to stimulate nuclear export.Recently emerged large amount of lncRNAs provoke further challenges in mechanisms of RNA localization.In contrast to mRNAs,lncRNAs do not encode protein and are reported to have fewer exons.The localization of lncRNAs was first believed mostly in the nucleus but gradually clarified as cytoplasmic dominantly.Analysis of Human and Drosophila subcellular compartments revealed that around?75%of lncRNAs enriched in cytoplasmic fractions.However,several intensively studied lncRNAs including XIST and MEG3 dominantly localize in the nucleus despite they are spliced,such dilemma can be explained by the presence of sequences/motifs in the lncRNAs that facilitate the retention in the nucleus.The localization and stability of XIST is dependent on sequences scattered in the RNA,with a 5' element vital to its correct localization and transcriptional silencing.For MEG3,nuclear retention element is mapped and the element can recruit U1 snRNP components to retain MEG3 in the nucleus.U1 snRNP interaction with RNA motif has also been linked with chromatin retention of other noncoding RNAs.In addition,a short pentamer AGCCC drives BORG localization while a longer repeating domain accomplishes same purpose for FIRRE.C-rich motifs derived from Alu repeats has been showed to govern lncRNA nuclear localization by binding to HNRNPK,C-rich nuclear enrichment pattern was also reported to be responsible for nuclear localization in several human lncRNAs.However,mechanism for nuclear export of intronless lncRNA is unknown at this stage.LncRNA localization has been strictly linked to their function at cellular level.Prevoiously NF-?B interacting lncRNA(NKILA)has been reported to accumulate in cytoplasm to blocks IKK phosphorylation motifs and inhibit phosphorylation of I?B in breast cancer cells.Which indicates the importance of NKILA nuclear export to perform its specific roles linked to cytoplasmic localization.Research MethodsNKILA(NR131157.1)is a cytoplasmic intronless lncRNA of 2,615 nucleotides.In this study,NKILA cytoplasmic localization in MCF7 cells was validated by Reverse Transcriptase Polymerase Chain Reaction(RT-PCR)after cell fractionation and Fluorescence In Situ Hybridization(FISH)was performed to visualize localization via microscope.To investigate sequence involved in NKILA localization,truncation and deletion constructs were designed FISH was performed for each construct,a 200-nucleotide sequence near 5' end was mapped in NKILA involved in its cytoplasmic accumulation and named as 'Cytoplasmic accumulation region' CAR-N.To validate CAR-N function in cytoplasmic accumulation of nuclear transcripts CAR-N was inserted at 5' of beta globin and GAS5 cDNA.To find CAR-N interacting proteins,CAR-N was in-vitro transcribed and biotin-streptavidin affinity purification was performed and followed by Mass Spectrometry(MS)analysis.Mass Spectrometry data was analyzed by Gene Ontology database and proteins categorized as "RNA Export/localization" were screened for effect on NKILA localization by using Small Interfering RNAs(siRNAs).From MS candidate proteins SRSF1 and SRSF7 were identified as trans factors recruited to CAR-N and mediated the export.SRSF1/7 binding site cluster was predicted in CAR-N by using bioinformatics tool and deletion of 55 nucleotides with the cluster of these binding sites lead to nuclear retention of NKILA reporter.Moreover,involvement of TREX/TAP pathway in NKILA export by siKD of UAP56,THOC2,AlyREF and TAP and effect of NKILA at reporter and endogenous level was also analyzed.In addition,effect of NKILA localization on migration of cells was analyzed by wound healing/scratch test after transfection of NKILA and NKILA lacking CAR-N in MCF-7 cells.ResultsTo confirm the localization of lncRNA NKILA at endogenous level,MCF7 cells were fractionated and nucleus and cytoplasmic separation was confirmed by western blot using UAP56 and tubulin as markers.RT-PCR was performed and NKILA RT-PCR band was predominantly cytoplasmic in MCF7 cells.NKILA sequence was cloned in pcDNA3 vector and transfected in MCF7 cells,RNA-FISH was performed to check localization of NKILA reporter,DAPI was used for nuclear staining.FISH signal was found predominantly in cytoplasm.To map cis-element involved in localization of NKILA,sequences of various length were deleted from reporter,transfected in MCF7 cells and FISH was employed to visualize localization.Truncation of 3' end didn't show any significant effect on cytoplasmic accumulation.However,a sequence of 200 nucleotides near 5' end(from nucleotide 250-450)was found to be responsible for NKILA cytoplasmic accumulation and it was termed as 'Cytoplasmic Accumulation Region CAR-N'.To test whether CAR-N functions in heterogeneous context,CAR-N was inserted at 5' end of nuclear transcripts of beta globin cDNA and GAS5 cDNA and transfected into MCF7 cells.RT-PCR of both transcripts showed no cryptic splicing in both transcripts.Nuclear transcripts of beta globin and GAS5 cDNA showed cytoplasmic accumulation after insertion of CAR-N as analyzed by RNA-FISH.To evaluate the role of protein in the export of NKILA,key components of TREX complex(THOC2,UAP56 and ALYREF)and mRNA export receptor TAP/NXF1 was depleted by respective siRNA then RNA-FISH and RT-PCR indicated that TREX complex was involved in export of NKILA.Depletion of UAP56,TAP lead to almost complete export block of NKILA while siThoc2 and siALYREF led to export block in?15%and 50%cells respectively.To investigate CAR-N interacting proteins,biotin labeled RNA pull down with CAR-N was performed and binding proteins were revealed by Mass Spectrometry analysis.Gene Ontology(GO)analysis categorized 13 proteins as "RNA Export "factors and knockdown of each of these proteins was done followed by RNA-FISH to find the effect on NKILA reporter localization.SRSF1 and SRSF7 knockdown significantly blocked nuclear export of NKILA at both reporter and endogenous level.To further evaluate the binding of SRSF1/7 to cis-element,protein binding sites were predicted within CAR-N and a cluster for SRSF1/7 sites were predicted and deletion of this cluster resulted in severe nuclear retention of NKILA.Additionally,full length NKILA suppressed migration of TGF-beta induced MCF7 cells while this effect was suppressed when CAR-N was deleted from NKILA,as assessed by wound healing analysis and western blot for expression of EMT markers E-Cadherin and Vimentin.ConclusionOverall this study showed that nuclear export of NKILA is sequence dependent process.A CAR was mapped in NKILA and named CAR-N,which functioned in both natural and heterologous contexts.Further,SRSF1 and SRSF7 as trans factors recruited to CAR-N and mediated the export.In addition,NKILA is also exported by TREX-TAP pathway and removal of CAR-N impaired its function to suppress cell migration.Overall,this study favor sequence dependent export of intronless lncRNAs via TREX-TAP pathway. |
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