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Warsaw University Faculty of Physics
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Role of 5' mRNA and 5' U snRNA cap structures in regulation of gene expression
 

Participation of MMG cap (m7GpppN) in formation of 48S translation initiation complex.
For details see: Gingras et al. "eIF4E initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation" in Annu. Rev. Biochem. 68, 913-963 (1999).

People involved:
Edward Darżynkiewicz
Janusz Stępiński
Jacek Jemielity
Joanna Żuberek
Ewa Grudzień
Katarzyna Kiraga-Motoszko
Magdalena Lewdorowicz
Dorota Haber
Marcin Kałek
Joanna Kowalska

Collaboration with (in Department of Biophysics):
Ryszard Stolarski
Jan Antosiewicz
Michał Dadlez

Further collaboration:
Marzena Jankowska-Anyszka, Faculty of Chemistry, Warsaw University
Zbigniew Wieczorek, Warmińsko-Mazurski University, Olsztyn
Andrzej Guranowski, Agriculture University, Poznań
Zbigniew Jedliński, Centre of Polymer Chemistry, PAS, Zabrze
Ryszard Kierzek, Institute of Bioorganic Chemistry, PAS, Poznań
Robert Rhoads, Louisiana State University, Shreveport, USA
Richard Davies, City University of New York, New York, USA
Nahum Sonenberg, McGill University, Montreal, Canada
Harri Lőnnberg, University of Turku, Turku, Finland
Michal Shapira, Ben Gurion University of The Negev, Israel
Iain Mattaj, European Molecular Biology Laboratory, Heidelberg, Germany
Stephen Cusack, European Molecular Biology Laboratory, Grenoble, France
 

 Keywords:
5' mRNA cap, (monometyloguanozyno kap; m7GpppG; MMG kap)
Eukariotic Initiation Factor 4E (eIF4E)
Cap Binding Complex (CBC)
m32,2,7GpppN (trimetylguanozyno kap; TMG kap)
U snRNA (małe j±drowe RNA)
Translacja
Splicing
 

Research interests:
Eukaryotic mRNA possesses on its 5'end a very specific structure, called cap, in which 7-metyloguanosine is connected with the next nucleoside through the non-typical for nucleic acids 5',5' triphosphate bond. Usually the cap structure is shown as m7GpppN or MMG cap (monomethyl guanosine cap). I nematodes, in addition to mRNAs capped with MMG cap, substantial amount of mRNAs possesses on their 5' ends trimethyl guanosine cap (TMG cap), in which 7-methyl guanosine is replaced by 2,2,7-trimethyl guanosine (m32,2,7GpppN). TMG cap is also present on 5' ends of the majority of U snRNAs.

MMG cap in mRNA plays an essential role in initiation of protein translation. It is recognized by eukaryotic protein factor eIF4E, which enables creation of initiation translation complex consisting of mRNA, a number of additional protein factors and a small ribosomal subunit 40S (see Scheme). MMG cap stimulates also splicing of pre-mRNA, and, to some extent, export of MMG-capped RNAs from the nucleus into the cytoplasm.

Laboratory of Gene Expression deals with synthesis of both natural as well as chemically modified cap analogues and their broad use in biochemical and biophysical research into molecular mechanisms of gene regulation. Specifically introduced chemical modifications on nucleic bases, ribofuranose rings and phosphate chain, provided over one hundred model cap analogues, many of which compete with mRNA for binding site in eIF4E thus inhibiting protein translation. Research into inhibition of protein translation by cap analogues enabled to define a number of structural cap parameters responsible for binding to eIF4E (f.e. see 1-3,11). We were the first to obtain synthetically TMG cap (m32,2,7GpppN) and its analogues (3), which were crucial to the discovery of signalling role of TMG cap in nuclear import of some U snRNAs (4,5), and to the discovery and initial characterisation of five eIF4E isoforms from nematode C.elegans (10,15,16).

Another application of MMG cap analogues and affinity resins based on them allowed to identify nuclear cap binding complex (CBC) and to prove its stimulating effect on pre-mRNA splicing (6,7). Currently, together with Professor Stolarski's team we are conducting research into interactions of eIF4E isoforms from C.elegans and CBC with model cap analogues. It is aimed to identify binding sites of specific protein factors to the cap structures.

Recently we have synthesized ARCAs: Anti Reverse Cap Analogues - dinucleotide cap analogues with chemically blocked 3' hydroxyl group in 7-methylguanosine (f.e. m73'OMeGpppG) (13). These analogues, thanks to their exclusively correct incorporation into RNA chain (in in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and dinucleotide cap analogue), have found broad application in preparation of synthetic RNA transcripts. mRNAs capped with ARCA proved to be twofold more effective templates in protein synthesis compared to their counterparts capped with traditional m7GpppG cap (13).

Our laboratory is also involved in obtaining the protein factors (wild type and mutants) essential to biophysical research into their interactions with cap structures (see f.e. 18).

The currently conducted projects involve: (a) chemical synthesis of oligonucleotides capped with natural and modified cap dinucleotides, (b) developing the efficient methodology of capping oligos on solid support, (c) synthesis of cap dinucleotides resistant to degradation by pyrophosphatases, (d) synthesis of new ARCAs with higher translational activity, (e) synthesis of new inhibitors of translation based on cap analogues that are able to cross the plasma membrane, in an attempt to target cap-dependent translation as an anticancer strategy, (f) investigation the role of eIF4E phosphorylation, (g) biophysical studies of cap analogs interactions with cap binding proteins (within close collaboration of Prof. R. Stolarski and Prof. J. Antosiewicz).

Selected publications:
  1. Darżynkiewicz E., Stępiński J., Ekiel I., Jin Y., Haber D., Sijuwade T., Tahara S.M., "ß-Globin mRNAs Capped with m7G, m2,7G or m2,2,7G Differ in Intrinsic Translation Efficiency" (1988), Nucleic Acids Res. 16, 8953-8962.
  2. Darżynkiewicz E., Stępiński J., Ekiel I., Goyer C., Sonenberg N., Temeriusz A., Jin Y., Sijuwade T., Haber D., Tahara S.M., "Inhibition of Eukaryotic Translation by Nucleoside 5'-Monophosphate Analogues of mRNA 5'-Cap: Changes in N7 Substituent Affect Analogue Activity" (1989), Biochemistry 28, 4771-4778.
  3. Darżynkiewicz E., Stępiński J., Tahara S. M., Stolarski R., Ekiel I., Haber D., Neuvonen K., Lehikoinen P., Labadi I., Lönnberg H., "Synthesis, Conformation and Hydrolytic Stability of P1, P3-dinucleoside Triphosphates Related to mRNA 5'-Cap, and Comparative Kinetic Studies on Their Nucleoside and Nucleoside Monophosphate Analogs" (1990), Nucleosides & Nucleotides 9, 599-618.
  4. Hamm J., Darżynkiewicz E., Tahara S. M., Mattaj I. W., "The Trimethylguanosine Cap Structure of U1 snRNA Is a Component of a Bipartite Nuclear Targeting Signal" (1990), Cell 62, 569-577.
  5. Fischer U., Darżynkiewicz E., Tahara S. M., Dathan N. A., Lührmann R., Mattaj I. W., "Diversity in the Signals Required for Nuclear Accumulation of U snRNPs and Variety in the Pathways of Nuclear Transport" (1991), J. Cell Biol. 113, 705-714.
  6. Izaurralde E., Stępiński J., Darżynkiewicz E., Mattaj I. W., "A Cap Binding Protein That May Mediate Nuclear Export of RNA Polymerase II-transcribed RNAs" (1992), J. Cell. Biol. 118, 1287-1295.
  7. Izaurralde E., Lewis J., McGuigan C., Jankowska M., Darżynkiewicz E., Mattaj I.W., "A Nuclear Cap Binding Protein Complex Involved in Pre-mRNA Splicing" (1994), Cell. 78, 657-668.
  8. Maroney P.A., Denker J.A., Darżynkiewicz E., Laneve R., Nilsen T.W., "Most mRNA in the Nematode Ascaris lumbricoides Are trans-spliced: A Role for Spliced Leader Addition in Translational Efficiency" (1995), RNA, 1, 714-723.
  9. Joshi B., Cai A-L., Keiper B. D., Minich W. M., Mendez R., Beach C. M., Stępiński J., Stolarski R., Darżynkiewicz E., Rhoads R. E, "Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E at Ser-209" (1995), J. Biol. Chem., 270, 14597-14603.
  10. Jankowska-Anyszka M., Lamphear B. J., Aamodt E. J., Harrington T., Darżynkiewicz E., Stolarski R., Rhoads R.E., "Multiple Isoforms of Eukaryotic Protein Synthesis Initiation Faktor 4E in Caenorhabditis elegans Can Distinguish between Mono- and Trimethylated mRNA Cap Structures" (1998), J. Biol. Chem. 273, 10538-10542.
  11. Cai A., Jankowska-Anyszka M., Centers A., Chlebicka L., Stępiński J., Stolarski R., Darżynkiewicz E., Rhoads R. E., "Quantitative Assessment of mRNA Cap Analogs as Inhibitors of Cell-Free Translation" (1999), Biochemistry, 38, 8538-8547.
  12. Lampio A., Ahola T., Darżynkiewicz E., Stępiński J., Jankowska-Anyszka M., Kaariainen L., "Guanosine Nucleotide Analogs as Inhibitors of Alphavirus mRNA Capping Enzyme" (1999), Antivirial Res., 42, 35-46.
  13. Stępiński J., Waddell C., Stolarski R., Darżynkiewicz E., Rhoads R.E.: "Synthesis and properties of mRNAs containing the novel 'anti-reverse' cap analogs 7-methyl-(3'-O-methyl)GpppG and 7-methyl-(3'-deoxy)GpppG", RNA 7 (2001) 1486-1495.
  14. NiedĽwiecka A., Marcotrigiano J., Stępiński J., Jankowska-Anyszka M., Wysłouch-Cieszyńska A., Dadlez M., Gingras A-C., Mak P., Darżynkiewicz E., Sonenberg N., Burley S.K., Stolarski R.: "Biophysical Studies of eIF4E Cap-binding Protein: Recognition of mRNA 5' Cap Structure and Synthetic Fragments of eIF4G and 4E-BP1 Proteins", J. Mol. Biol. 319 (2002) 615-635.
  15. H. Miyoshi, D.S. Dwyer, B. D. Keiper, M. Jankowska-Anyszka, E. Darżynkiewicz, R.E. Rhoads: "Discrimination between mono- and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E", EMBO J. 21 (2002) 4680-4690.
  16. A. Stachelska, Z. Wieczorek, K. Ruszczyńska, R. Stolarski, M. Pietrzak, B. J. Lamphear, R. E. Rhoads, E. Darżynkiewicz, M. Jankowska-Anyszka: "Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues", Acta Biochim. Pol. 49 (2002) 671-682.
  17. A. NiedĽwiecka, J. Stępiński, E. Darżynkiewicz, N. Sonenberg, R. Stolarski: "Positive heat capacity change upon specific binding of translation itiation factor eIF4E to mRNA 5' cap", Biochemistry 41 (2002) 12140-12148.
  18. J. Żuberek, A. Wysłouch-Cieszyńska, A. NiedĽwiecka, M. Dadlez, J. Stępiński, W. Augustyniak, A.-Cl. Gingras, Z. Zhang, S. K. Burley, N. Sonenberg, R. Stolarski, E. Darżynkiewicz: "Phosphorylation of eIF4E attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein", RNA 9 (2003) 52-61.

Supported by:
 
GRANT International Research Scholars Program, HHMI #55005604 pt. "Synthetic 5' mRNA Cap Analogues: Tools to Search for Gene-Regulatory Mechanisms" (2006-2010)
 
GRANT MNiI 2 P04A 006 28 pt. "Struktura i funkcja kapu-4 u wczesnych eukariotów - trypanosomatydów, jego chemiczne analogi jako potencjalne leki przeciwko Leishmanii" (2005 - 2008)
 
Grant PBZ-KBN 059/T09/10: "Synthesis of mono-, di-, tetra- i oligonucleotide 5' mRNA cap analogues - potential anticancer drugs and tools for research into molecular biology" (2001-2005)