While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.
References
[1]
Frydmann J (2001) Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem 70: 603–647.
[2]
Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295: 1852–1858.
[3]
Kerner MJ, Naylor DJ, Ishihama Y, Maier T, Chang HC, et al. (2005) Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122: 209–220.
[4]
Vorderwülbecke S, Kramer G, Merz F, Kurz TA, Rauch T, et al. (2004) Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK. FEBS Lett 559: 181–187.
[5]
Ullers RS, Luirink J, Harms N, Schwager F, Georgopoulos C, et al. (2004) SecB is a bona fide generalized chaperone in Escherichia coli. Proc Natl Acad Sci U S A 101: 7583–7588.
[6]
Wall JG, Plückthun A (1995) Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6: 507–516.
[7]
Davis GD, Elisee C, Newham DM, Harrison RG (1999) New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol Bioeng 65: 382–388.
[8]
Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8: 1668–1674.
[9]
Braun P, LaBaer J (2003) High throughput protein production for functional proteomics. Trends Biotechnol 21: 383–388.
[10]
Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17: 353–358.
[11]
Bukau B, Horwich AL (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92: 351–366.
[12]
Chiti F, Calamai M, Taddei N, Stefani M, Ramponi G, et al. (2002) Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Proc Natl Acad Sci U S A 99: 16419–16426.
[13]
Uversky VN, Gillespie JR, Fink AL (2000) Why are “natively unfolded” proteins unstructured under physiologic conditions? Proteins 41: 415–427.
[14]
Otzen DE, Kristensen O, Oliveberg M (2000) Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: a structural clue to amyloid assembly. Proc Natl Acad Sci U S A 97: 9907–9912.
[15]
Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424: 805–808.
[16]
Chen J, Skehel JJ, Wiley DC (1998) A polar octapeptide fused to the N-terminal fusion peptide solubilizes the influenza virus HA2 subunit ectodomain. Biochemistry 37: 13643–13649.
[17]
Zhang YB, Howitt J, McCorkle S, Lawrence P, Springer K, et al. (2004) Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expr Purif 36: 207–216.
[18]
Kim CW, Han KS, Ryu KS, Kim BH, Kim KH, et al. (2007) N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers. Protein Sci 16: 635–643.
[19]
Su Y, Zou Z, Feng S, Zhou P, Cao L (2007) The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli. J Biotechnol 129: 373–382.
[20]
Rentzeperis D, Jonsson T, Sauer RT (1999) Acceleration of the refolding of Arc repressor by nucleic acids and other polyanions. Nat Struct Biol 6: 569–573.
[21]
Frankel AD, Smith CA (1998) Induced folding in RNA-protein recognition: more than a simple molecular handshake. Cell 92: 149–151.
[22]
Das B, Chattopadhyay S, Bera AK, Dasgupta C (1996) In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA. Eur J Biochem 235: 613–621.
[23]
Kudlicki W, Coffman A, Kramer G, Hardesty B (1997) Ribosomes and ribosomal RNA as chaperones for folding of proteins. Fold Des 2: 101–108.
[24]
Moore PB (2001) The ribosome at atomic resolution. Biochemistry 40: 3243–3250.
[25]
Brevet A, Chen J, Lévque F, Blanquet S, Plateau P (1995) Comparison of the enzymatic properties of the two Escherichia coli lysyl-tRNA synthetase species. J Biol Chem 270: 14439–14444.
[26]
Elton D, Medcalf L, Bishop K, Harrison D, Digard P (1999) Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding. J Virol 73: 7357–7367.
[27]
Miller JD, Bernstein HD, Walter P (1994) Interaction of E. coli Ffh/4.5S ribonucleoprotein and FtsY mimics that of mammalian signal recognition particle and its receptor. Nature 367: 657–659.
[28]
Frank DN, Pace NR (1998) Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu Rev Biochem 67: 153–180.
[29]
Korber P, Zander T, Herschlag D, Bardwell JC (1999) A new heat shock protein that binds nucleic acids. J Biol Chem 274: 249–256.
[30]
Onesti S, Desogus G, Brevet A, Chen J, Plateau P, et al. (2000) Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding. Biochemistry 39: 12853–12861.
[31]
Commans S, Plateau P, Blanquet S, Dardel F (1995) Solution structure of the anticodon-binding domain of Escherichia coli lysyl-tRNA synthetase and studies of its interaction with tRNALys. J Mol Biol 253: 100–113.
[32]
Wang CC, Morales AJ, Schimmel P (2000) Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase. J Biol Chem 275: 17180–17186.
[33]
Kim JM, Sohn HY, Yoon SY, Oh JH, Yang JO, et al. (2005) Identification of gastric cancer-related genes using a cDNA microarray containing novel expressed sequence tags expressed in gastric cancer cells. Clin Cancer Res 11: 473–482.
[34]
Kim NS, Hahn Y, Oh JH, Lee JY, Oh KJ, et al. (2004) Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags. Genomics 83: 1024–1045.
[35]
The information about tested proteins is available at the web site of the ‘Functional Analysis of Human Genome’ in part of 21C Frontier R&D Project in Korea (http://kugi.kribb.re.kr:8080/genbank/ind？ex.jsp).
[36]
Nasoff M, Bergseid M, Hoeffler JP, Heyman JA (2000) High-throughput expression of fusion proteins. Methods Enzymol 328: 515–529.
[37]
Endo Y, Sawasaki T (2006) Cell-free expression systems for eukaryotic protein production. Curr Opin Biotechnol 17: 373–380.
[38]
Tickle I, Sharff A, Vinkovic M, Yon J, Jhoti H (2004) High-throughput protein crystallography and drug discovery. Chem Soc Rev 33: 558–565.
[39]
Dyson HJ, Wright PE (2002) Coupling of folding and binding for unstructured proteins. Curr Opin Struct Biol 12: 54–60.
[40]
Wittung-Stafshede P (2002) Role of cofactors in protein folding. Acc Chem Res 35: 201–208.
[41]
Alexandrescu AT, Jaravine VA, Dames SA, Lamour FP (1999) NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding. J Mol Biol 289: 1041–1054.
Ellis RJ, Hartl FU (1999) Principles of protein folding in the cellular environment. Curr Opin Struct Biol 9: 102–110.
[44]
Feldman DE, Frydman J (2000) Protein folding in vivo: the importance of molecular chaperones. Curr Opin Struct Biol 10: 26–33.
[45]
Craig EA, Eisenman HC, Hundley HA (2003) Ribosome-tethered molecular chaperones: the first line of defense against protein misfolding? Curr Opin Microbiol 6: 157–162.
[46]
Lee J, Choi SI, Jang JS, Jang K, Moon JW, et al. (1999) Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer. Biotechnol Prog 15: 884–890.
[47]
Makino Y, Amada K, Taguchi H, Yoshida M (1997) Chaperonin-mediated folding of green fluorescent protein. J Biol Chem 272: 12468–12474.
[48]
Weinstein Y, Ihle JN, Lavu S, Reddy EP (1986) Truncation of the c-myb gene by a retroviral integration in an interleukin 3-dependent myeloid leukemia cell line. Proc Natl Acad Sci U S A 83: 5010–5014.
[49]
Kaminska M, Denniziak M, Kerjan P, Barciszewski J, Mirande M (1999) A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation. EMBO J 19: 6908–6917.
