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© David Marcey, 2001
II. The U1A Protein-U1 snRNA Complex
III. U1A Protein-U1A pre-mRNA Binding
Note: This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.
Pre-mRNA splicing reactions are catalyzed by the concerted action of 5 small nuclear ribonucleoproteins (snRNPs). The U1 snRNP consists of a 164 nucleotide RNA (U1 snRNA), eight core proteins shared by all snRNPs, and three U1-specific proteins.
In one of the first steps in intron removal, the U1A snRNP binds to the 5' splice junction through pairing between the 5' end of the U1 snRNA and the first few nucleotides at the 5' end of the intron to be removed. Other domains of the extensively folded U1 snRNA are involved with binding both core proteins and U1-specific proteins, including U1A, shown at left complexed with a 21 nucleotide RNA representing hairpin II of the U1 snRNA (Oubridge, et al., 1994).
Structural analysis will be important in elucidating the catalytic mechanisms of complex snRNP machines. This exhibit, based on the work of Oubridge, et al. (1994) and Jovine, et al. (1996), examines the binding of U1A protein to both the U1 snRNA and the 3' untranslated region of the mRNA that encodes the U1A protein.
The loop of the U1 snRNA hairpin II comprises ten nucleotides , and projects perpendicularly from the double helical stem. Seven loop nucleotides, AUUGCAC (residues 6-12), are conserved in hairpin IV of U2 snRNA and in loops found in the 3' untranslated region of the pre-mRNA encoding U1A (see below). The remaining three nucleotides in the loop make no significant contact with U1A and likely serve as a linker between the conserved AUUGCAC sequence and the stem.
The AUUGCAC stretch is observed to make extensive contact with a four-stranded b-sheet of U1A . U1A contains an RNP motif found in over 150 different RNA binding proteins. This motif of ~80 amino acids has two highly conserved stretches, RNP1 and RNP2, which are adjacent on two central strands of the U1A b-sheet . The C-terminal region of U1A stretches across the b-sheet, ending in a short a-helix .
A protein loop between b-strands 2 and 3 of the sheet pokes into the RNA hairpin and disrupts potential base pairing of loop nucleotides . The hydrogen bond acceptor and donor atoms of the splayed nucleotides are thus made available for numerous direct or H2O-mediated hydrogen bonds with U1A.
The RNP1 and RNP2 sequences and surrounding regions contain residues that are key for bonding to the AUUGCAC residues. Arginine 52, the first amino acid of RNP1, protrudes from the b2-b3 loop and forms hydrogen bonds with atoms of RNA residues A6 and G16, helping to position the RNA appropriately . The ability of U1A to bind RNA is completely abolished if arg52 is replaced with glutamine (Nagai, et al., 1990). Asparagines 15 and 16 of RNP2 bond with G9 and U8 . Glutamate 19, adjacent to RNP2, contacts both U7 and G9 .
Residues in the C-terminal region of U1A also participate in H-bonding with the RNA loop . The C-terminal peptide parallels the RNA backbone, with many main-chain carbonyls and amides interacting with C10, A11, and C12.
In addition to the extensive H bonding just described, hydrophobic stacking interactions between nitrogenous bases and amino acid side chains are observed. C10 stacks on tyrosine 13 of RNP2 and A11 and C12 are sandwiched between the side chains of phenylalanine 56 (RNP1) and aspartate 92 from the C-teminus .
U1A protein negatively regulates its own expression by binding the U1A 3' untranslated region of its pre-mRNA, thereby inhibiting polyadenylation. The structure shown at left is a theoretical model of a U1A protein dimer (residues 2-97) complexed with the U1A pre-mRNA 3' UTR (Jovine, et al., 1996). The complex shows that the protein chains are opposed face to face, with the extensively looped 3' UTR nestled on their surface . The two protein chains are held together by hydrophobic interactions at their interface .
Interestingly, the same chemical strategy is used by U1A to bind the 3' UTR and the U1 snRNA (see previous section). In particular, note the following conserved interactions:
Arginine 52, the first amino acid of RNP1, protrudes from the b2-b3 loop and interacts with nucleotides near the base of the RNA loops.
H bonding and aromatic stacking interactions between key residues in the RNP1 and RNP2 U1A regions and the AUUGCAC (AUUGUAC) sequences in two internal RNA loops that lie upstream of the polyadenylation signal.
Jovine, L., Oubridge, C., Avis, J., and K. Nagai (1996). Two structurally different RNA molecules are bound by the splicesomal protein U1A using the same recognition strategy. Structure 4: 621.
Nagai, K., Oubridge, C., Jessen, T.H., Li, J., and P.R. Evans. (1990) Crystal structure of the RNA binding domain of the U1 small nuclear ribonucleoprotein A. Nature 348: 515-520.
Oubridge, C., Ito, N., Evans,
P.R., Teo, C., and K. Nagai. (1994) Crystal structure at 1.92 angstrom resolution
of the RNA-binding domain of the U1A spliceosomal protein complexed with an
RNA hairpin. Nature 372: 432-438.
For Further Reading
Boelens, W.C., Jansen, E.J.R., van Venrooij, W.J., Stripecke, R., Mattaj, I.W., and S.I. Gunderson. (1993) The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell 72: 881-892.
Burd, C.G., and G. Dreyfuss. (1994) Conserved structures and diversity of functions of RNA-binding proteins. Science 265: 615-621.
Hall, K.B. (1994) Interaction of RNA hairpins with the human U1A N-terminal RNA binding domain. Biochemistry 33: 10076-10088.
Howe, P.W.A, Nagai, K.,
Neuhaus, D., and G. Varani. (1994) NMR studies of U1 snRNA recognition by the
N-terminal RNP domain of the human U1A protein. EMBO Journal 13:
Laird-Offringa, I.A., and J.G. Belasco. (1995) Analysis of RNA-binding proteins by in vitro genetic selection: Identification of an amino acid residue important for locking U1A onto its RNA target. PNAS 92: 11859-11863.
Mattaj, I.W. (1993) RNA recognition: A family matter? Cell 73: 837-840.