Research of the Wahle group
Our research interests
3´-processing of mRNA
In eukaryotic cells, the transcripts generated by RNA polymerase II are converted into mature mRNAs by three major processing steps: addition of the 5‘ cap, splicing of introns and processing of the 3‘ end. The latter event is one focus of our research.
3‘ processing consists of two steps, the endonucleolytic cleavage of a phosphodiester bond in the pre-mRNA, followed by the addition of a poly(A) tail to the upstream cleavage product containing the coding sequences. Functions of the poly(A) tail include mRNA export to the cytoplasm, a stimulation of translation and participation in the regulation of mRNA stability. 3‘ processing is directed by several sequences in the pre-mRNA, including, in animal cells, the well-known AAUAAA sequence some 20 nucleotides upstream of the cleavage and polyadenylation site.
Although 3‘ end formation appears to require only two catalytic functions, an endonuclease and a poly(A) polymerase, the processing machinery is complex, consisting of at least fifteen polypeptides. One key player is Cleavage and Polyadenylation Specificity Factor (CPSF), a heterooligomer that specifically binds to the AAUAAA sequence and is required for cleavage as well as for polyadenylation. Cleavage also requires three heterooligomeric cleavage factors and poly(A) polymerase. Although this enzyme, which is responsible for the addition of the poly(A) tail, plays a catalytic role only in the second part of the 3` processing reaction, it is already present in the complex that performs the cleavage of the pre-mRNA. Only CPSF and poly(A) polymerase are required for polyadenylation; the cleavage factors are dispensable. However, for efficient poly(A) synthesis, an additional protein, the nuclear Poly(A) Binding Protein 1 (PABPN1) is also needed. Through direct interactions with poly(A) polymerase, CPSF and PABPN1 tether the enzyme to the RNA. As a result, polyadenylation becomes processive, i. e. poly(A) polymerase can synthesize a complete poly(A) tail without dissociating from the RNA. Interestingly, this processive reaction terminates once the physiologically correct poly(A) tail length of approximately 250 nucleotides has been reached. Whereas processive polyadenylation requires the simultaneous interaction of poly(A) polymerase with both PABPN1 and CPSF, the latter contact is disrupted when the tail reaches a length of ~250 nucleotides. PABPN1 is responsible for measuring the length of the tail and for the CPSF – poly(A) polymerase being maintained only during the addition of the first ~250 nucleotides.
We are currently trying to obtain more structural information on the proteins involved in polyadenylation and their interactions.
Selected publications:
Wahle, E. (1991) A novel poly(A)-binding protein acts as a specificity factor in the second phase of poly(A) tail synthesis. Cell 66, 759-768
Bienroth, S., Keller, W., Wahle, E. (1993) Assembly of a processive polyadenylation complex. EMBO J. 12, 585-594
Wahle, E. (1995) Poly(A) tail length control is caused by termination of processive synthesis. J. Biol. Chem. 270, 2800-2808
Nemeth, A., Krause, S., Blank, D., Jenny, A., Jenö, P., Lustig, A., Wahle, E. (1995) Isolation of genomic and cDNA clones encoding bovine poly(A) binding protein II. Nucleic Acids Res. 23, 4034-4041
Keller, R. W., Kühn, U., Aragon, M., Bornikova, L., Wahle, E., Bear, D. G. (2000) The nuclear poly(A) binding protein 2, PABP2, forms an oligomeric particle covering the length of the poly(A) tail. J. Mol. Biol. 297, 569-583
Kühn, U., Nemeth, A., Meyer, S., Wahle, E. (2003) The RNA binding domains of the nuclear poly(A) binding protein. J. Biol. Chem. 278, 16916-16925.
Kerwitz, Y., Kühn, U., Scheuermann, T., Lilie, H., Schwarz, E., Wahle, E. (2003) Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA. EMBO J. 22, 3705-3714.
Kühn, U., Gündel, M., Knoth, A., Kerwitz, Y., Rüdel. S., Wahle, E. (2009) Poly(A) tail length is controlled by the nuclear poly(A) binding protein regulating the interaction between poly(A) polymerase and the cleavage and polyadenylation specificity factor. J. Biol. Chem., in press.
Mechanism and function of PABPN1 modification by protein arginine methyltransferases
The nuclear poly(A) binding protein (PABPN1) plays a role in polyadenylation of pre-mRNA in the cell nucleus (see above). A C-terminal domain of 49 amino acids contains 13 arginine residues, all of which are asymmetrically dimethylated. Among the many arginine residues outside the C-terminal domain, only two are partially methylated. Asymmetric dimethylarginine was initially found mainly in the so-called RGG domain of RNA binding proteins, but was later identified also in proteins involved in signal transduction, transcription factors and histones H3 and H4. We have identified protein arginine methyltransferases PRMT1, -3 und -6 as the enzymes responsible for the methylation of PABPN1. Even though these enzymes are oligomeric, and methylated arginines are clustered in most known substrate proteins, the reaction mechanism is distributive. In the case of PABPN1, substrate specificity is determined by local amino acid sequences rather than by neighboring protein domains, and the PRMTs operate on their own rather than as catalytic subunits of larger assemblies. We are trying to characterize their substrate specificities, and we are also interested in the biological function of arginine methylation, paying particular attention to a possible role in intracellular trafficking of PABPN1.
Publications:
Smith, J. J., Rücknagel, P., Schierhorn, A., Nemeth, A., Tang, J., Linder, M., Herschman, H. R., Wahle, E. (1999) Unusual sites of arginine methylation in poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3. J. Biol. Chem. 274, 13229-13234
Ostareck-Lederer, A.*, Ostareck, D. H., Rücknagel, K. P., Schierhorn, A., Moritz, B., Hüttelmaier, S., Flach, N., Handoko, L., Wahle, E. (2006) Asymmetric arginine dimethylation of hnRNP K by PRMT1 inhibits its interaction with c-Src. J. Biol. Chem. 281, 11115-11125. *corresponding author
Fronz, K., Otto, S., Kölbel, K., Kühn, U., Friedrich, H., Schierhorn, A., Beck-Sickinger, A. G., Ostareck-Lederer, A., Wahle, E. (2008) Promiscuous modification of the nuclear poly(A) binding protein by multiple protein arginine methyl transferases does not affect the aggregation behavior. J. Biol. Chem. 283, 20408-20420.
Kölbel, K., Ihling, C., Bellmann-Sickert, K., Neundorf, I., Beck-Sickinger, A. G., Sinz, A., Kühn, U., Wahle, E. (2009) Type I arginine methyl transferases PRMT1 and 3 act distributively. J. Biol. Chem. 284, 8274-8282.
Mechanism and regulation of mRNA decay
Gene expression is regulated not only at the level of transcription and translation, but also at the level of mRNA stability. The abundance of mRNAs with a short half-life can be regulated very rapidly. Unstable mRNAs include many of biological and medical interest, like those encoding cytokines or several transcription factors like c-fos or c-myc.
In most cases, the first step in the degradation of mRNA is an exonucleolytic removal of the poly(A) tail. Certain destabilizing elements in the mRNA determine the rate of deadenylation as well as the overall rate of mRNA degradation, and investigations of these sequences have shown that deadenylation is frequently the rate-determining step of mRNA decay.
We have purified and cloned PARN, a poly(A)-specific 3` exonuclease from mammalian tissue, which is stimulated by a cap structure at the 5’ end of the substrate. In collaboration with Mike Wormington (Charlottesville, Virginia, USA) we have shown that the frog homolog of PARN is responsible for a deadenylation reaction that occurs during the meiotic maturation of Xenopus oocytes and is used for the purpose of translational regulation.
We are interested in the role of this enzyme in somatic cells.
The most important and generally conserved mRNA deadenylase is the so-called CCR4-NOT complex, which consists of several subunits including two 3’ exonucleases, CCR4 and CAF1 (or POP2). We have shown that this complex is responsible for the deadenylation of bulk mRNA and of the hsp70 mRNA in Drosophila. We are currently characterizing the subunit composition of the complex and the functions of individual subunits.
Two general pathways of mRNA decay have been characterized in yeast. Both start with deadenylation. In one pathway, the next step is hydrolysis of the 5’ cap, followed by 5’ exonuclease degradation of the RNA. In the alternative pathway, the RNA is degraded from the 3’ end without prior hydrolysis of the cap. We have analyzed the decay pathway of the hsp70 mRNA in Drosophila and found that the major pathway of its decay is deadenylation followed by cap hydrolysis and 5’ decay. Degradation from the 3’ end is a minor pathway.
We are currently characterizing intermediates of this pathway in more detail. Both deadenylation and cap hydrolysis are retarded during heat shock. Rapid decay of the hsp70 mRNA is governed by sequences in the 3’ UTR, but not by the type of AU-rich elements that direct the rapid decay of many mammalian mRNAs.
We have also developed a cell-free system, derived from Drosophila embryos, which reproduces both the deadenylation and the translational repression of the nanos mRNA seen in vivo. Deadenylation as well as translational repression depend on the so-called Smaug response elements (SREs) in the 3’ UTR. Interestingly, rapid deadenylation is also ATP-dependent. We are interested in studying the mechanism of both reactions, SRE-dependent deadenylation and translational repression.
Publications:
Körner, C., Wahle, E. (1997) Poly(A) tail shortening by a mammalian poly(A)-specific 3’-exoribonuclease. J. Biol. Chem. 272, 10448-10456
Körner, C. G., Wormington, M., Muckenthaler, M., Schneider, S., Dehlin, E., Wahle, E. (1998) The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes. EMBO J. 17, 5427-5437
Dehlin, E., Wormington, M., Körner, C. G., Wahle, E. (2000) Cap-dependent deadenylation of mRNA. EMBO J. 19, 1079-1086
van Dijk, E., Cougot, N., Meyer, S., Babajko, S., Wahle, E., Séraphin, B. (2002) The human Dcp2 protein: a catalytically active mRNA decapping enzyme localized in specific cytoplasmic structures. EMBO J. 21, 6915-6924.
Temme, C., Zaessinger, S., Meyer, S., Simonelig, M., Wahle, E. (2004) A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J. 23, 2862-2871.
Meyer, S., Temme, C., Wahle, E. (2004) Messenger RNA turnover in eukaryotes: Pathways and enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 197-216.
Wu, M., Reuter, M., Lilie, H., Liu, Y., Wahle, E., Song, H. (2005) Structural insight into poly(A) binding and catalytic mechanism of human PARN. EMBO J. 24, 4082-4093.
Jeske, M., Meyer, S., Temme, C., Freudenreich, D., Wahle, E. (2006) Rapid ATP-dependent deadenylation of nanos mRNA in a cell-free system from Drosophila embryos. J. Biol. Chem. 281, 25124-25133.
Bönisch, C., Temme, C., Moritz, B, Wahle, E. (2007) Degradation of hsp70 and other mRNAs in Drosophila via the 5'-3' pathway and its regulation by heat shock. J. Biol. Chem., 282, 21818 - 21828.
Jeske, M., Wahle, E. (2008) Cell-free deadenylation assays using Drosophila embryo extracts. Methods Enzymol. 448, 107-118.