Department for Molecular Structural Biology

Nucleo cytoplasmic trafficking

d. Biogenesis of UsnRNPs: An Im- and Export story

In eukaryotic cells, mRNAs are generally transcribed as pre-mRNAs in which the information for the protein sequence is contained in so-called exons, coding regions of variable length, and intervening non-coding regions the introns. Thus, the newly synthesized pre-mRNA is composed of an array of exons and introns. In order to obtain meaningful genetic information leading to functional protein, the introns have to be removed, a process occuring before export of the mRNA to the cytoplasm where translation by the ribosome takes place. The excision of an intron and fusion of the adjacent exons is achieved by the spliceosome, a supramolecular, highly dynamic, ribonucleoprotein (RNP) complex.

Essential components of the major spliceosome are the five Uridyl-rich small nuclear RNPs (UsnRNPs) U1, U2, U4, U5 and U6 and several non-snRNP proteins. Each UsnRNP is composed of a specific UsnRNA and a set of seven common proteins, the Sm proteins, for U1, U2, U4, U5 or highly homologous proteins to those seven, the Lsm proteins, for U6. Additionally each UsnRNP acquires a subset of particle specific proteins.



NT-AD-Fig8sFig.1. Biogenesis of UsnRNPs.
[Click for a larger image]

The biogenesis of spliceosomal UsnRNPs in higher eukaryotes requires a cytoplasmic maturation step (Fig. 1). Thus, after transcription and initial processing within the nucleus the snRNAs U1, U2, U4 and U5 are exported to the cytoplasm in an m7G-Cap dependent manner by the CRM1 dependent pathway as well as the proteins PHAX, CBP20 and 80 as mediators. In the cytoplasm these UsnRNAs specifically associate with seven Sm-proteins that form a doughnut-shaped UsnRNP core structure. This assembly, its formation mediated by the SMN complex, is a prerequisite for the hypermethylation of the m7G-cap to the 2,2,7-trimethylguanosine (m3G)-cap (Step 1). Snurportin1 specifically recognizes this m3G-cap and in concert with other import factors facilitates the import of core UsnRNPs into the nucleus (Step 2/3). Here the complex disassembles in an ordered fashion and SPN1 is transported back into the cytoplasm in a CRM1/RanGTP dependent manner (Step 4)



For a more detailed information on the biogenesis of UsnRNPs and the underlying transport processes see:

  • Dickmanns, A. (2009). Import and Export of UsnRNPs. Nuclear Transport; Edited by Ralph H. Kehlenbach, Landes Bioscience

  • Dickmanns, A. and Ficner, R. (2005). Role of the 5'-cap in the biogenesis of spliceosomal snRNPs. Topics in Current Genetics 12, 179-204.



  • Recent research has focused on the structural requirements for the interaction of the following steps in the biogenesis (numbers according to the numbers in Fig. 1):



    1. cap hypermethylation

    The trimethylguanosine synthase TGS1 comprises a region resembling the canonical methyltransferase domain for substrate and ligand (SAM) binding. Structure determination of this domain revealed a fold structurally similar to the core domain of methyltransferases, but activity tests showed no activity (Fig. 2). Stepwise addition of residues at the N-terminus to the canonical methyltransferase domain led to more and more active forms of TGS1 (see activity test, Fig. 2, middle). Crystal structure analysis of the active form revealed an additional N-terminal domain, its correct formation essential for binding to both ligand and substrate (Fig. 2).



    NT-AD-Fig9mFig.2. TGS1 requires an N-terminal domain additionally to the MTase domain for activity.The active fold is dramatically altered (blue region, left image) in comparison to the inactive form (red strands, right panel)



    References

  • Monecke, T., Dickmanns, A. and Ficner, R. (2009). Erkennung des RNA-5`-caps durch Proteine. Biospektrum 07.09, 720-725.

  • Monecke, T., Dickmanns, A., and Ficner, R. (2009). Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1.Nucleic Acids Res. 37, 3865-3877. [Abstract]; PDB: [3GDH]

  • Monecke,T., Dickmanns, A., Strasser A., and Ficner R. (2009) . Structure analysis of the conserved methyltransferase domain of human trimethylguanosine synthase TGS1.Acta Cryst. D65, 332-338. [Abstract]; PDB: [3EGI]


  • 2. Recognition of the core UsnRNP by SPN1

    The nuclear import adaptor SPN1 specifically recognizes the hypermethylated 5'-cap by its cap-binding domain (Fig. 3). In contrast to other cap binding proteins it requires the cap-nucleotide and the first nucleotide of the UsnRNA for binding (Fig. 4).



    NT-AD-Fig10mFig.3. Import adaptor Snurportin1. Left: SPN1 domain architecture and overall structure of the cap-binding domain. Right: The trimethylated cap and the first nucleotide are both required for binding to SPN1. The two nucleotides are stacked between a tryptophan (276) on one side and a leucine (104) at the other side of the pocket. A second tryptophan (107) shields the cap to the surrounding water.



    Fig4_Snurportin_CBP20Fig. 4. Comparison of the Snurportin 1 cap-binding pocket with the m7G-cap-binding pockets of Cap binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and the viral nucleoside 2´-O-methyltransferase (VP39). Side chains of the residues interacting with the caps are depicted in ball-and-stick mode, the atoms of the caps and the interacting side chains are colored as follows: nitrogen atoms in blue, oxygens in red and phosphorus atoms in green. Carbons of the respective proteins are shown in grey, but the carbon atoms of the dinucleotide in orange. In all presented cases, the residues stacking the bases and those forming hydrogen bonds (dashed gray lines) with the cap-bases are indicated.




    References

  • Kühn-Hölsken, E., Lenz, C., Dickmanns, A., Hsiao, H.-H., Richter, F.M., Kastner, B., Ficner, R., and Urlaub, H. (2010). Mapping the binding site of Snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry. Nucleic Acids Res. 38(16):5581-93. [Abstract]

  • Götte, M., Stumpe, M.C., Ficner, R. and Grubmüller, H. (2009). Molecular determinants of snurportin1 ligand affinity and structural response upon binding..Biophys. J. 97, 581-589. [Abstract]

  • Strasser, A., Dickmanns, A., Lührmann, R. and Ficner, R. (2005). Structural basis for m3G-cap mediated nuclear import of spliceosomal UsnRNPs by snurportin1. EMBO J. 24, 2235-2243. [Abstract]; PDB:[1XK5]

  • Strasser, A., Dickmanns, A., Schmidt, U., Penka, E., Urlaub, H., Sekine, R., Lührmann, R. and Ficner, R. (2004). Purification, crystallization and preliminary crystallographic data of the m3G cap-binding domain of human snRNP import factor snuportin1. Acta Cryst. D60, 1628-1631. [Abstract]


  • 3. Import properties of SPN1 and Binding to Importin-ß

    Importin-ß binding (IBB)-domain structure
    The N-terminal IBB-domain of Snurportin1 binds to the C-terminal region of Importin-ß similar to the IBB domain (Fig. 5) of a second well studied adaptor Importin-α. Surprisingly differences in the IBB domain sequence result in differences in the disassembly of the import complex. Whereas RanGTP binding to Importin-ß bound to Importin-α requires RanGTP for release from the NPC and disassembly, Importin-ß bound to SPN1 requires RanGTP only fort the latter.

    NT-AD-Fig11Fig.5. IBB-domain of SPN1 bound to Importin ß. The crystal structure of the IBB-domain from SPN1 bound to Importin-ß exhibits an extended conformation similar to the one observed in Fig 3 (purple helix) in contrast to the Importin-ß bound to the IBB-domain of Importin-α.



    References

  • Wohlwend, D., Strasser, A., Dickmanns, A., and Ficner R. (2007). Structural Basis for RanGTP Independent Entry of Spliceosomal U snRNPs into the Nucleus.J Mol Biol. 2007 Dec 7;374(4):1129-38. [Abstract]; PDB: [2QNA]


  • 4. Recycling of SPN1

    Once SPN1 has released the core UsnRNPs into the nucleus, most likely within the Cajal Bodies, it is recycled into the cytoplasm by the CRM1-dependent pathway. The binding has been shown to occur in a co-operative manner together with RanGTP.

  • Read More: CRM1 dependent export


  • References

  • Monecke, T., Güttler, T., Neumann, P., Dickmanns, A., Görlich, D., and Ficner, R. (2009). Crystal structure of the nuclear export receptor CRM1 in complex with snurportin1 and RanGTP.Science 324, 1087-1091. [Abstract]; PDB: [3GJX]

  • Güttler T., Madl T., Neumann P., Deichsel D., Corsini L., Monecke T., Ficner R., Sattler M. & Görlich D. (2010). NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nature Struct. Mol. Biol. (11):1367-76. [Abstract]; PDB: [3NBY][3NBZ][3NC0][3NC1]

  • Monecke, T., Haselbach, D., Voß, B., Russek, A., Neumann, P., Thomson, E., Hurt, E., Zachariae, U., Stark, H., Grubmüller, H., Dickmanns, A. and Ficner, R. (2013). Structural basis for cooperativity of CRM1 export complex formation. PNAS USA. 110, 960-965. [Abstract]; [Publication]; PDB: [4FGV], [4HZK], EMDB: [EMD-2110], [EMD-2111]

  • Dölker, N., Blanchet, C.E., Voß, B., Haselbach, D., Kappel , C., Monecke, T., Svergun, D.I., Stark, H., Ficner, R., Zachariae, U., Grubmüller, H. and Dickmanns, A. (2013) . Structural determinants and mechanism of mammalian CRM1 allostery. Structure, 21:1350-1360. [Abstract]; EMDB: [EMD-2274] [EMD-5564]

  • Monecke, T., Dickmanns, A. and Ficner, R. (2014). Allosteric control of the exportin CRM1 unraveled by crystal structure analysis FEBS Journal, accepted [Abstract]