Abstract

Structure and function of A1 heterogeneous nuclear ribonuclear protein (hnRNP) will be studied. This is an abundant protein that binds pre-mRNA. There is disagreement in the literature over whether or not it binds sequence specifically. One goal of this project is to measure the sequence specificity of binding using an equilibrium titration method developed in this lab. The effects of posttranslational modifications, such as methylation and phosphorylation on binding, will also be studied. """"""""A major effort....will be to solve the structure of an A1:RNA complex."""""""" Both x-ray crystallography and NMR methods will be tried. The effect on binding of the length of linker arms connecting two RNA binding domains will also be studied and compared with theory developed by Crothers. A yeast two-hybrid system will be used to identify proteins that interact with A1. The sequence specificity of RNA binding and the proteins that bind should provide insight into the function of A1, including how it modulates selection of alternative 5'-splice sites. Some conclusions may be relevant to the more than 100 other RNA binding proteins that use the same RNA binding motif as A1.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031539-14
Application #
2391920
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1983-01-01
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
14
Fiscal Year
1997
Total Cost
Indirect Cost

  Institution

Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520

  Publications

Kim, S; Merrill, B M; Rajpurohit, R et al. (1997) Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif. Biochemistry 36:5185-92
Abdul-Manan, N; O'Malley, S M; Williams, K R (1996) Origins of binding specificity of the A1 heterogeneous nuclear ribonucleoprotein. Biochemistry 35:3545-54
Abdul-Manan, N; Williams, K R (1996) hnRNP A1 binds promiscuously to oligoribonucleotides: utilization of random and homo-oligonucleotides to discriminate sequence from base-specific binding. Nucleic Acids Res 24:4063-70
O'Malley, S M; Sattar, A K; Williams, K R et al. (1995) Mutagenesis of the COOH-terminal region of bacteriophage T4 regA protein. J Biol Chem 270:5107-14
Amrute, S B; Abdul-Manan, Z; Pandey, V et al. (1994) Purification and nucleic acid binding properties of a fragment of type C1/C2 heterogeneous nuclear ribonucleoprotein from thymic nuclear extracts. Biochemistry 33:8282-91
Shamoo, Y; Abdul-Manan, N; Patten, A M et al. (1994) Both RNA-binding domains in heterogenous nuclear ribonucleoprotein A1 contribute toward single-stranded-RNA binding. Biochemistry 33:8272-81
Garrett, D S; Lodi, P J; Shamoo, Y et al. (1994) Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy. Biochemistry 33:2852-8
Nadler, S G; Merrill, B M; Roberts, W J et al. (1991) Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions. Biochemistry 30:2968-76
Merrill, B M; Barnett, S F; LeStourgeon, W M et al. (1989) Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles. Nucleic Acids Res 17:8441-9
Roberts, W J; Pan, T; Elliott, J I et al. (1989) p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39. Biochemistry 28:10043-7

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