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The Hilda Pomeraniec Professorial Chair of Organic Chemistry76100 Rehovot, Israel telephone: +972 (8) 934-2509 e-mail: j.sperling@weizmann.ac.il |
Members of the group:
Eyal Kamhi eyal.kamhi@weizmann.ac.il Gideon Kass gidi.kass@weizmann.ac.il Tali Dadosh tali.dadosh@weizmann.ac.il Yael Hacham yael.hacham@weizmann.ac.il
Research in our Group
The pre-mRNA processing machine: Functional characterization and 3-D image reconstruction.
Nuclear RNA transcripts of split genes are spliced prior to their export to the cytoplasm while they are packaged in a multicomponent large nuclear ribonucleoprotein (lnRNP) particle. Our laboratory was the first to isolate, visualize by electron microscopy, and biochemically characterize these lnRNP particles. An isolated lnRNP particle is a huge macromolecular assembly that contains, in addition to pre-mRNA, all known components required for its splicing, as well as other RNA processing components required for capping, polyadenylation and editing of pre-mRNAs. Hence, the lnRNP particle can be regarded as representing the nuclear RNA processing machine.
The objective of our research is the elucidation of the function and structure of the lnRNP particles, thereby achieving a better understanding of cellular key regulatory mechanisms pertaining to RNA processing, and particularly to pre-mRNA splicing.
In our structural work we determined the mass of the lnRNP particle by scanning transmission electron microscopy (STEM), and used electron tomography from two-dimensional TEM images to reconstruct its three-dimensional structure. This model has further been used to explain how the reading-frame of an mRNA can be recognized in the cell nucleus prior to splicing.
To enable the visualization by EM of RNA in the context of the lnRNP particle, we have developed chemical procedures whereby nucleic acids (RNA and DNA) can be decorated with sold clusters. This research has recently been expanded to the field of microelectronics in collaboration with colleagues from the Department of Condensed Matter Physics.
Recognition of open reading frames in pre-mRNAs within the cell nucleus
Pre-mRNA splicing involves recognition of a consensus sequence at the 5' splice site. However, only some of the many potential sites that conform to the consensus are true ones, while the majority remain silent and are not normally used for splicing. We noticed that in most cases he utilization of such a latent intronic 5'splice site for splicing would introduce an in frame stop codon into the resultant mRNAs. This suggested a link between splice site selection and maintenance of an open reading frame, which is manifested by suppressing splicing events that could lead to the inclusion of pre-mature termination codons in mRNAs. We have experimentally verified this suppression of splicing (SOS) idea and found that splicing events at latent sites are indeed activated by mutations that eliminate the interfering stop codons. Our findings imply that the reading frame of mRNAs can be recognized in the cell nucleus prior to splicing, presumably by a surveillance mechanism that resides in the pre-mRNA processing machine. This nuclear scanning mechanism is highly important for accurate gene expression, as perturbations that would lead to splicing at latent sites are expected to introduce in frame stop codons into the majority of mRNAs.
Click here for list of all publications since 2000 (including DOIs) [Submit additions and corrections]
Selected publications
E. Kamhi, G. Yahalom, G. Kass, Y. Hacham, R. Sperling, and J. Sperling, "AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing", Nucleic Acids Research 34, 3421-3433 (2006). [Read online]
Azubel, M., Habib, N, Sperling, R. and Sperling, J. Native spliceosomes assemble with Pre-mRNA to form supraspliceosomes. Journal of Molecular Biology 356, 955-966 (2006). [Read online]
Dadosh T, Gordin Y, Krahne R, Khivrich I, Mahalu D, Frydman V, Sperling J, Yacoby A, Bar-Joseph I, "Measurement of the conductance of single conjugated molecules", Nature 436, 677-680 (2005); erratum 436, 1200 (2005). [Read online; Online erratum]
Azubel, M., Wolf, S. G., Sperling, J. and Sperling, R. Three-dimensional structure of the native spliceosome by cryo-electron microscopy. Mol. Cell 15, 833-839 (2004). [Read online]
Wachtel, C., Li, B., Sperling, J. and Sperling, R. Stop codon-mediated suppression of splicing is a novel nuclear scanning mechanism not affected by elements of protein synthesis and NMD. RNA 10, 1740-1750 (2004). [Read online]
Miriami, E., Sperling, R., Sperling, J. and Motro, U., Regulation of splicing: the importance of being translatable, RNA 10, 1--4 (2004). [Read online; Online Supplementary Material]
Krahne R, Dadosh T, Gordin Y, Yacoby A, Shtrikman H, Mahalu D, Sperling J , Bar-Joseph I . Nanoparticles and nanogaps: controlled positioning and fabrication, Physica E 17, 498-502 (2003). [read online]Uhle, S., Medalia, O., Waldron, R., Dumdey, R., Henklein, P., Bech-Otschir, D., Huang, X., Berse, M., Sperling, J., Schade, R., and Dubiel, W. Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome. EMBO Journal 22, 1302-1312 (2003). [Read online]
Krahne, R., Yacoby, A., Shtrikman, H., Bar-Joseph, I., Dadosh, T., and Sperling, J. Fabrication of nanoscale gaps in integrated circuits. Applied Physics Letters 81, 730-732 (2002). [Read online]
Li, B., Wachtel, C., Miriami, E., Yahalom, G., Friedlander, G., Sharon, G., Sperling, R., and Sperling, J. Stop codons affect 5' splice site selection by surveillance of splicing. Proc. Natl. Acad. Sci. USA 99, 5277-5282 (2002). [read online]
Miriami, E., Motro, U., Sperling, J., and Sperling, R. Conservation of an open-reading frame as an element affecting 5' splice site selection. J. Struct. Biol. 140, 116-122 (2002). [Read online]
Medalia, O., Englander, J., Guckenberger, R., and Sperling, J. AFM Imaging in Solution of Protein-DNA Complexes Formed on DNA Anchored to a Gold Surface. Ultramicroscopy 90, 103-112 (2002). [read online]
Medalia, O., Typke, D., Hegerl, R., Angenitzki, M., Sperling, J., and Sperling, R. Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine. J. Struct. Biol. 138, 74-84 (2002). [Read online]
Raitskin, O., Angenitzki, M., Sperling, J., and Sperling, R. Large nuclear RNP particles-the nuclear pre-mRNA processing machine. J. Struct. Biol. 140, 123-130 (2002). [Read online]
N.J. Duker, J. Sperling, K.J. Soprano, D.P. Druin, A. Davis and R. Ashworth. beta-amyloid protein induces the formation of purine dimers in cellular DNA. J. Cell. Biochem. 81, 393-400 (2001)[read online].
O. Raitskin, D. S. Cho, J. Sperling, K. Nishikura and R. Sperling RNA editing activity is associated with splicing factors in lnRNP particles - The Nuclear pre-mRNA processing machinery. Proc. Natl. Acad. Sci. USA 98, 6571-6576 (2001).[read online]
H. Holzl, B. Kapelari, J. Kellermann, E. Seemuller, M. Sumegi, A. Udvardy, O. Medalia, J. Sperling, S. A. Muller, A. Engel and W. Baumeister. The regulatory complex of drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme. J. Cell Biol. 150, 119-130 (2000).[read online]
O. Medalia, M. Heim, R. Guckenberger, R. Sperling and J. Sperling. Gold-tagged RNA-A probe for macromolecular assemblies. J. Struct. Biol. 127, 113-119 (1999).[read online]
S. Müller, B. Wolpensinger, M. Angenitzki, A. Engel, J. Sperling and R. Sperling. A supraspliceosome model for large nuclear ribonucleoprotein particles based on mass determinations by scanning transmission electron microscopy. J. Mol. Biol. 283, 383-394 (1998).[read online]
O. Medalia, A.J. Koster, A. Tocilij, M. Angenitzki, J. Sperling, Z. Berkovitch-Yellin and R. Sperling. Automated electron tomography of large nuclear RNP (InRNP) particles - The naturally assembled complexes of precursor messenger RNA and splicing factors. J. Struct. Biol. 120, 228-236 (1997).[read online]
R. Sperling, A. J. Koster, C. Melamed-Bessudo, A. Rubinstein, M. Angenitzki, Z. Berkovitch-Yellin and J. Sperling, Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography. J. Mol. Biol. 267, 570-583 (1997).[read online]
S. Yitzhaki, E. Miriami, J. Sperling and R. Sperling, Phosphorylated SR proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles. Proc. Natl. Acad. Sci. USA 93, 8830-8835 (1996).[read online]
E. Miriami, M. Angenitzki, R. Sperling and J. Sperling, Magnesium cations are required for the association of U snRNPs and SR proteins with pre-mRNA in 200S large nuclear ribonucleoprotein (lnRNP) particles. J. Mol. Biol. 246, 254-263 (1995).[read online]E. Miriami, J. Sperling and R. Sperling, Heat shock affects 5' splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in lnRNP particles. Nucleic Acids Res. 22, 3084-3091 (1994).