Skip to main content
SpringerLink
Log in

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion.

Abstract

The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3′ splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins’ Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ATP:

Adenosine triphosphate

FLIP:

Fluorescence loss in photobleaching

FRAP:

Fluorescence recovery after photobleaching

GFP:

Green fluorescence protein

LMB:

Leptomycin B

NaN3:

Sodium azide

Pre-mRNA:

Precursor messenger RNA

SF1:

Splicing factor 1

snRNP:

Small nuclear ribonucleoproteins

U2AFs:

U2 snRNP auxiliary splicing factor

NES:

Nuclear export signal

References

  • Abel S, Theologis A (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5:421–427

    Article  CAS  PubMed  Google Scholar 

  • Ali GS, Reddy ASN (2006) ATP, phosphorylation and transcription regulate the mobility of plant splicing factors. J Cell Sci 119:3527–3538

    Article  CAS  PubMed  Google Scholar 

  • Ali GS, Reddy ASN (2008a) Regulation of alternative splicing of pre-mRNAs by stresses. Curr Top Microbiol Immunol 326:257–275

    CAS  PubMed  Google Scholar 

  • Ali GS, Reddy ASN (2008b) Spatiotemporal organization of pre-mRNA splicing proteins in plants. Curr Top Microbiol Immunol 326:103–118

    CAS  PubMed  Google Scholar 

  • Ali GS, Golovkin M, Reddy ASN (2003) Nuclear localization and in vivo dynamics of a plant-specific serine/arginine-rich protein. Plant J 36:883–893

    Article  CAS  PubMed  Google Scholar 

  • Barta A, Marquez Y, Brown JWS (2012) Challenges in plant alternative splicing. In: Smith CWJ, Lührmann R (eds) Alternative Pre-mRNA splicing: theory and protocols. Wiley-VCH Verlag, Weinheim, pp 79–89

    Chapter  Google Scholar 

  • Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C (2002) Membrane protein transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching. Plant Cell 14:1293–1309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Burge CB, Tuschl T, Sharp PA (1999) Splicing of precursors to mRNAs by the spliceosome. In: Gesteland RF, Cech TR, Atkins JF (eds) The RNA world. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 525–560

    Google Scholar 

  • Carmo-Fonseca M (2002) The contribution of nuclear compartmentalisation to gene regulation. Cell 108:513–521

    Article  CAS  PubMed  Google Scholar 

  • Chusainow J, Ajuh PM, Trinkle-Mulcahy L, Sleeman JE, Ellenberg J, Lamond AI (2005) FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35. RNA 11:1201–1214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Docquier S, Tillemans V, Deltour R, Motte P (2004) Nuclear bodies and compartmentalisation of pre-mRNA splicing factors in higher plants. Chromosoma 112:255–266

    Article  CAS  PubMed  Google Scholar 

  • Domon C, Lorković ZJ, Valcarcel J, Filipowicz W (1998) Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants. J Biol Chem 273:34603–34610

    Article  CAS  PubMed  Google Scholar 

  • Dong XH, Biswas A, Süel KE, Jackson LK, Martinez R, Gu HM et al (2009) Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature 458:1136–1141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fang Y, Hearn S, Spector DL (2004) Tissue-specific expression and dynamic organisation of SR splicing factors in Arabidopsis. Mol Biol Cell 15:2664–2673

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gama-Carvalho M, Carmo-Fonseca M (2001) The rules and roles of nucleocytoplasmic shuttling proteins. FEBS Lett 498:157–163

    Article  CAS  PubMed  Google Scholar 

  • Gama-Carvalho M, Carvalho MP, Kehlebbach A, Valcárcel J, Carmo-Fonseca M (2001) Nucleocytoplasmic shuttling of heterodimer splicing factor U2AF. J Biol Chem 16:13104–13112

    Article  Google Scholar 

  • Hackmann A, Wu H, Schneider UM, Meyer K, Jung K, Krebber H (2014) Quality control of spliced mRNAs requires the shuttling SR proteins Gbp2 and Hrb1. Nat Commun 5:3123

    Article  PubMed  Google Scholar 

  • Iida K, Seki M, Sakurai T, Satou M, Akiyama K et al (2004) Genome-wide analysis of alternative pre-mRNA splicing in Arabidopsis thaliana based on full-length cDNA sequences. Nucleic Acids Res 32:5096–5103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jang YH, Park HY, Lee KC, May PT, Kim SK, Suh MC et al (2014) The Arabidopsis homolog of splicing factor SF1 is involved in the alternative splicing of pre-mRNA and development in Arabidopsis thaliana. Plant J 78:591–603

    Article  CAS  PubMed  Google Scholar 

  • Jurica MS, Moore MJ (2003) Pre-mRNA splicing: awash in a sea of proteins. Mol Cell 12:5–14

    Article  CAS  PubMed  Google Scholar 

  • Koroleva OA, Clader G, Pendle AF, Kim SH, Lewandowska D, Simpson CG et al (2009) Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia. Plant Cell 21:1592–1606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kudo N, Wolff B, Sekimoto T, Schreiner EP, Yoneda Y, Yanagida M et al (1998) Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp Cell Res 242:540–547

    Article  CAS  PubMed  Google Scholar 

  • Lamond AI, Earnshaw WC (1998) Structure and function in the nucleus. Science 280:547–553

    Article  CAS  PubMed  Google Scholar 

  • Lamond AI, Spector DL (2003) Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol 4:605–612

    Article  CAS  PubMed  Google Scholar 

  • Lorković ZJ, Barta A (2004) Compartmentalization of the splicing machinery in plant cell nuclei. Trends Plant Sci 9:565–568

    Article  PubMed  Google Scholar 

  • Maldonado-Bonilla LD (2014) Composition and function of P bodies in Arabidopsis thaliana. Front Plant Sci 5:201. doi:10.3389/fpls

    Article  PubMed  PubMed Central  Google Scholar 

  • Maroney PA, Romfo CM, Nilsen TW (2000) Functional recognition of the 5′ splice site by U4/U6·U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol Cell 6:317–328

    Article  CAS  PubMed  Google Scholar 

  • Misteli T (2000) Different site, different splice. Nat Cell Biol 2:E98–E100

    Article  CAS  PubMed  Google Scholar 

  • Misteli T (2001) Protein dynamics: implications for nuclear architecture and gene expression. Science 291:843–847

    Article  CAS  PubMed  Google Scholar 

  • Misteli T, Spector DL (1998) The cellular organization of gene expression. Curr Opin Cell Biol 10:322–331

    Article  Google Scholar 

  • Misteli T, Spector DL (1999) RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. Mol Cell 3:697–705

    Article  CAS  PubMed  Google Scholar 

  • Misteli T, Caseras JF, Spector DL (1997) The dynamics of a pre-mRNA splicing factor in living cells. Nature 387:523–527

    Article  CAS  PubMed  Google Scholar 

  • Moore MJ (2000) Intron recognition comes of AGe. Nat Struct Biol 7:14–16

    Article  CAS  PubMed  Google Scholar 

  • Patel AA, Steitz JA (2003) Splicing double: insights from the second spliceosome. Nat Rev Mol Cell Biol 4:960–970

    Article  CAS  PubMed  Google Scholar 

  • Proudfoot NJ, Furger A, Dye MJ (2002) Integrating mRNA processing with transcription. Cell 108:501–512

    Article  CAS  PubMed  Google Scholar 

  • Rausin G, Tillemans V, Stankovic N, Hanikenne M, Motte P (2010) Dynamic nucleocytoplasmic shuttling of an Arabidopsis SR splicing factor: role of the RNA-binding domains. Plant Physiol 153:273–284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reddy ASN (2001) Nuclear pre-mRNA splicing in plants. Crit Rev Plant Sci 20:523–571

    Article  CAS  Google Scholar 

  • Reddy ASN (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58:267–294

    Article  CAS  PubMed  Google Scholar 

  • Rino J, Carvalho T, Braga J, Desterro JMP, Luhrmann R, Carmo-Fonseca M (2007) A stochastic view of spliceosome assembly and recycling in the nucleus. PLoS Comput Biol 3:2019–2031

    Article  CAS  PubMed  Google Scholar 

  • Rino J, Desterro JMP, Pacheco TR, Gadella TW, Carmo-Fonseca M (2008) Splicing factors SF1 and U2AF associate in extraspliceosomal complexes. Mol Cell Biol 28:3045–3057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ruskin B, Zamore PD, Green MR (1988) A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell 52:207–219

    Article  CAS  PubMed  Google Scholar 

  • Spector DL (1993) Macromolecular domains within the cell nucleus. Annu Rev Cell Biol 9:265–315

    Article  CAS  PubMed  Google Scholar 

  • Tillemans V, Dispa L, Remacle C, Collinge M, Motte P (2005) Functional distribution and dynamics of Arabidopsis SR splicing factors in living plant cells. Plant J 41:567–582

    Article  CAS  PubMed  Google Scholar 

  • Tillemans V, Leponce I, Rausin G, Dispa L, Motte P (2006) Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors. Plant Cell 18:3218–3234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Twyffels L, Gueydan C, Kruys V (2011) Shuttling SR proteins: more than splicing factors. FEBS J 278:3246–3255

    Article  CAS  PubMed  Google Scholar 

  • Wang BB, Brendel V (2006) Molecular characterization and phylogeny of U2AF35 homologs in plants. Plant Physiol 140:624–636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weigel D, Glazebrook J (2002) Arabidopsis. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Zaidi SK, Young DW, Choi JY, Pratap J, Javed A, Montecino M et al (2005) The dynamic organisation of gene-regulatory machinery in nuclear microenvironments. EMBO Rep 6:128–133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zamore PD, Green MR (1989) Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci USA 86:9243–9247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang XN, Mount SM (2009) Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development. Plant Physiol 150:1450–1458

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education (to J.-K. Kim), and by a Korea University Grant (to J.-K. Kim).

Author information

Author notes

  1. Hyo-Young Park and Keh Chien Lee contributed equally to this work.

Authors and Affiliations

  1. Department of Life Sciences, Korea University, Anam-dong 5 ga, Seongbuk-gu, Seoul, 02841, Republic of Korea

    Hyo-Young Park, Keh Chien Lee, Yun Hee Jang, May Phyo Thu & Jeong-Kook Kim

  2. Division of Biological and Environmental Sciences and Engineering, Center for Desert Agriculture, King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia

    Soon-Kap Kim

  3. Department of Life Sciences, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-Si, Jeollabuk-do, 54896, Republic of Korea

    Jeong Hwan Lee

Authors
  1. Hyo-Young Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Keh Chien Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yun Hee Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Soon-Kap Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. May Phyo Thu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeong Hwan Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jeong-Kook Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jeong Hwan Lee or Jeong-Kook Kim.

Ethics declarations

Conflict of interest

The authors have no conflicts of interest to declare.

Additional information

Communicated by Inhwan Hwang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

299_2017_2142_MOESM1_ESM.avi

Figure S1. Time-lapse movies derived from the FLIP experiment shown in Fig. 5.Table S1. Primers used in this study. (AVI 24841 kb)

Supplementary material 2 (AVI 33984 kb)

Supplementary material 3 (DOCX 14 kb)

Supplementary material 4 (AVI 26524 kb)

Supplementary material 5 (AVI 24516 kb)

Supplementary material 6 (AVI 26298 kb)

Supplementary material 7 (AVI 26043 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Park, HY., Lee, K.C., Jang, Y.H. et al. The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. Plant Cell Rep 36, 1113–1123 (2017). https://doi.org/10.1007/s00299-017-2142-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-017-2142-z

Keywords

Search

Navigation