The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

[1]  E. Conti,et al.  The structure of the mRNA export factor TAP reveals a cis arrangement of a non‐canonical RNP domain and an LRR domain , 2000, The EMBO journal.

[2]  C. Burd,et al.  hnRNP proteins and the biogenesis of mRNA. , 1993, Annual review of biochemistry.

[3]  E. Conti,et al.  Nucleocytoplasmic transport enters the atomic age. , 2001, Current opinion in cell biology.

[4]  S. Piñol-Roma.  Association of nonribosomal nucleolar proteins in ribonucleoprotein complexes during interphase and mitosis. , 1999, Molecular biology of the cell.

[5]  D. E. Wolf,et al.  Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Lamond,et al.  Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus , 1995, The Journal of cell biology.

[7]  A. Lustig,et al.  Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides. , 1993, The Journal of biological chemistry.

[8]  D. Newmeyer,et al.  Nuclear import can be separated into distinct steps in vitro: Nuclear pore binding and translocation , 1988, Cell.

[9]  A. Beyer,et al.  Identification and characterization of the packaging proteins of core 40S hnRNP particles , 1977, Cell.

[10]  M. Carmo-Fonseca,et al.  Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA. , 2000, Journal of cell science.

[11]  W. Ansorge,et al.  Microinjection of Anti-coilin Antibodies Affects the Structure of Coiled Bodies , 1998, The Journal of cell biology.

[12]  G. Blobel,et al.  Gene gating: a hypothesis. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Hauber,et al.  Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)–And Constitutive Transport Element (Cte)–Containing Retroviral Rnas , 2001, The Journal of cell biology.

[14]  P. L. Taylor,et al.  The Meaning of Nucleocytoplasmic Transport , 1996, Molecular Biology Intelligence Unit.

[15]  W. Richardson,et al.  Nuclear protein migration involves two steps: Rapid binding at the nuclear envelope followed by slower translocation through nuclear pores , 1988, Cell.

[16]  G. Dreyfuss,et al.  Immunological methods for purification and characterization of heterogeneous nuclear ribonucleoprotein particles. , 1990, Methods in enzymology.

[17]  B. Séraphin,et al.  Dbp5, a DEAD‐box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p , 1999, The EMBO journal.

[18]  L. Maquat,et al.  Quality Control of mRNA Function , 2001, Cell.

[19]  E. Wahle.  A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation , 1991, Cell.

[20]  S. Belikov,et al.  Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3'-end formation and excision of the 3'-terminal intron. , 1998, Genes & development.

[21]  K. Nasmyth,et al.  Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. , 1997, Science.

[22]  J. Neefjes,et al.  From fixed to FRAP: measuring protein mobility and activity in living cells , 2001, Nature Cell Biology.

[23]  Jason R. Swedlow,et al.  Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast , 1997, Nature.

[24]  E. Hurt,et al.  The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans , 2000, Nature.

[25]  C. Peterson,et al.  Actin-related proteins (Arps): conformational switches for chromatin-remodeling machines? , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[26]  B. Daneholt,et al.  Actin Bound to the Heterogeneous Nuclear Ribonucleoprotein Hrp36 Is Associated with Balbiani Ring mRNA from the Gene to Polysomes , 2001, The Journal of cell biology.

[27]  G. Dreyfuss,et al.  Transport of Proteins and RNAs in and out of the Nucleus , 1999, Cell.

[28]  T. Pederson.  Proteins associated with heterogeneous nuclear RNA in eukaryotic cells. , 1974, Journal of molecular biology.

[29]  J. Besag.  On the Statistical Analysis of Dirty Pictures , 1986 .

[30]  A. Verkman,et al.  Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus , 1997, The Journal of cell biology.

[31]  P. Silver,et al.  Messenger RNAs are recruited for nuclear export during transcription. , 2001, Genes & development.

[32]  S. Masich,et al.  The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles. , 1999, Experimental cell research.

[33]  E. Stelzer,et al.  Photobleaching GFP reveals protein dynamics inside live cells. , 1999, Trends in cell biology.

[34]  T. Deerinck,et al.  In vivo analysis of the stability and transport of nuclear poly(A)+ RNA , 1994, The Journal of cell biology.

[35]  R. Singer,et al.  Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells , 1999, Current Biology.

[36]  N. Proudfoot,et al.  Terminal exon definition occurs cotranscriptionally and promotes termination of RNA polymerase II. , 1999, Molecular cell.

[37]  U. Kutay,et al.  Deciphering the cellular pathway for transport of poly(A)-binding protein II. , 2000, RNA.

[38]  E. Wahle,et al.  Immunodetection of poly(A) binding protein II in the cell nucleus. , 1994, Experimental cell research.

[39]  T. Misteli,et al.  High mobility of proteins in the mammalian cell nucleus , 2000, Nature.

[40]  U. Kutay,et al.  The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. , 2000, RNA.

[41]  Petar M. Djuric,et al.  Unsupervised vector image segmentation by a tree structure-ICM algorithm , 1996, IEEE Trans. Medical Imaging.

[42]  B. Daneholt,et al.  The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm , 2002, The EMBO journal.

[43]  A. Lustig,et al.  Isolation of genomic and cDNA clones encoding bovine poly(A) binding protein II. , 1995, Nucleic acids research.

[44]  M. Rode,et al.  Overexpression of TAP/p15 Heterodimers Bypasses Nuclear Retention and Stimulates Nuclear mRNA Export* , 2001, The Journal of Biological Chemistry.

[45]  J. Darnell,et al.  hnRNA in HeLa cells: Distribution of transcript sizes estimated from nascent molecule profile , 1976, Cell.

[46]  J. Shabanowitz,et al.  A myosin I isoform in the nucleus. , 2000, Science.

[47]  G. Georgiev.  3 – Precursor of mRNA (Pre-mRNA) and Ribonucleoprotein Particles Containing Pre-mRNA , 1974 .

[48]  U. Kutay,et al.  Transport between the cell nucleus and the cytoplasm. , 1999, Annual review of cell and developmental biology.

[49]  J. Yong,et al.  Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. , 2000, Molecular cell.

[50]  K Ribbeck,et al.  Kinetic analysis of translocation through nuclear pore complexes , 2001, The EMBO journal.

[51]  B. Fahrenkrog,et al.  The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG‐nucleoporin Rip1p, the DEAD‐box protein Rat8p/Dbp5p and a new protein Ymr255p , 1999, The EMBO journal.

[52]  J. Burke,et al.  A relative weighting method for estimating parameters and variances in multiple data sets , 1996 .

[53]  W. Webb,et al.  Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. , 1976, Biophysical journal.

[54]  N. Proudfoot,et al.  Multiple Transcript Cleavage Precedes Polymerase Release in Termination by RNA Polymerase II , 2001, Cell.