- Email: [email protected]
A protein splice-junction motif in hedgehog family proteins
P 0TEI E!IEICE 0TIF
T~BS 2 0 - APRIL 1 9 9 5 11 E~en, ~. L L. et aL (1993) Gone132, 143-148 ./.2 Klenk, H-P.and Doo~itt~e,W. E {1994) Curt. BioL 4, 920-922 13 OuzounisC. and Sander, C. (1992) Ce~ 71, 189-199 i 4 Marsh, T. L., Reich, C. L, White~ock,R. B.and OIsen, G. J. {1994) Prec. Nat/Acad. ScL USA 9!, 4180-4.!84 15 Row~ands,T., Baumann, P. and Jackson, S. P. (1994) Science264,1326=1329
Protein splicing is a recently discovered, complex post-translational modification that includes two concerted proteolytic cleavages and one ligaiion reacUon, and produces two distinct, functional proteins from a single polypeptide ~-s. By analog,:, with introns and exons in RNA precursors, the internal protein that is excised from the translation product is called intein, whereas the two terminal portions, when ligated, form a fusion protein called extein ~. So far, the intein-extein organization has been observed in about ten proteins from yeast, bacteria and Archaea; in most of these cases, the inteins are inserted within or near nucleotide-binding domains ~,5.Protein splicing is thought to be an autocatalytic process, since it can occur in heterologous systems; for example, intein-contalning proteins from Mycobacterium and Thermococcus have been synthesized in Escherichia coilTM, and protein splicing has been demonstrated with the purified inteincontaining DNA polymerase from
~tOCOCCUS9. Comparative analysis of the amino acid sequences of spliced proteins has revealed several conserved motifs (Ref. 5 and references therein). With only one exception, all the inteins are related to the yeast homing endonuclease (HO) and contain two dodecapeptide motifs that are typical of members of a large superfamily of endonucleases, mostly encoded by mobile group I introns ~,m. However, the endonuclease activity is not required for protein splicino . Two other conserved motifs are found around the intein--extein splice junctions ~,s.The amino-terminal (N~)junction motif is found in those of the inteins that have cysteine at the splice site, while inteins that have serine in this position show very little, if any, sequence conservation. The carboxy-terminal (C') junction motif is conserved in all known inteins and contains invariant histidine and asparagine residues 4,s. Here, I describe a
16 Langer, O.and ZiHig, W. (1993) Nucleic Adds Res. 21, 2251-2251 17 Kaine, ~. P., Mehr, O.J. and Woese, C. R. (1994) Prec. Natl Acad. ScL USA 91, 3854-3856 ~80uzounis, C. A., Kyrpides, N. C. and Sander,C. Nucleic Acids Res. {in press) ~90uzounis, C. A. and ~yrpides, N. C. (1994) J. Mol. Evol. 39, 101-104 20 Woese,C. R. (1987) MicmbieLRev. 51, 221-271
Eurcpean Molecular 8iolo~ ~ ~:,:~ory, Postfach 102209, D-69102 Hei~lberg, Germany (current address: A~Center, SR~~nternat~onaL 333 RavenswoodAve,M~nloPa~, CA 94025-34~o3. USA).
protein sptice-~unction motif in the hedgehog (lift) family of eukaryot~c developmental regulatory proteins, and discuss the possibility that it is involved in autoproteolysis and perhaps splicing of these proteins. When the amino acid sequences of the known intein-containing proteins were compared with a nonredundant database @~R)using the BLASTP program ~, moderate similarity (score of 76, probability of occurring by chance approximately 0.08) was observed between the N' splice junction motif of Mycobacterium Ppsl protein s and a segment of HH, a developmental regulatory protein from Xenopus laevis.
Somewhat lower scores were observed between the splice-~undion motifs from Ppsl and yeast vacuolar ATPases, and HH-related proteins from Drosophila to mammals. The region of similarity included the junction motif, and the splice-site cysteine was conserved in all the sequences (Fig. 1). The block of aligned cysteine-contalnlng splice junctions was converted into a positiondependent weight matrb~, which was used to search the NR iteratively ~. The HH family proteins were the only sequences selected; the probability of the HH sequences matching the motif by chance, given the size of the database, was computed to be between 10-4 and 10~.
institute of ~olecu~ar 8iolog~~nd ~iotechnelogyo PO ~o~ 1527, GR-71110oHerak~ior~,Gree~.
| s.on...G/CU ..... u.U..go...U..U..C,d.~ consensus 1 HH_DRO~ 249 81SSHVHG/CFTPESTALLE°SGVRKPLGELSIgDRVL 186 SONHH mou 190 SLAVRAGG/CrPGNATVRLR-SGEP~GL~LHRGD~A~L 173 SOR~H ~ e ~ 185 S%~SGG/CrPGSALVSLO-DGGQ~AVKDT~PGDKVL 193 219 HH Xenla 190 S V A ~ S G G / C F P A G A R V M V E - ~ G T K A V K D ~ R P ~ D R V L Ppsl Mlep~ 193 NTA%~SGG/CLTADARZ~-G~GLVSZADVQP~DEV~ ~ATA_CA~TR 275 S D V X ~ Y V ~ / C ~ T ~ G T ~ - D ~ A D K S Z E S Z E V G D K ~ VATA~YEAST 275 $ D A Z ~ G / C ~ A K G T ~ - D ~ S ~ R C Z E N Z E V G ~ K ~
640 778 761
Pyrsp 398 N I V Y L D F I / C H P A D T ~ - G K G I I N Z S E V O E G D Y V L
135
POL
R E C A ~ Y C L E 198 E E Z G V J ~ G / ~ S T R V T ~ - D G S T E K Z G K Z V N N E M D V REC~CTU ~43 R ~ N K / C L A E G T R Z F D P ~ I T G T T H R I E D V V D G R ~ P X . . . n . . . g / ~ ..... u.u..go...u..U..gd.uU consensus 2
473 510
Rgure1 The N' splice-junction motif in the hedgehogfamily proteins and in intein¢ontaining proteins. The alignment was generated by a combination of CAP and MoST programs~, and a gap was introduced to achieve the optimal alignment of the RECA_MYCTUsequence. The protein sequences were from SWISS-PROTor from GenPept if a SWISS-PROTentry was unavailable. Hedgehogfamily proteins: HH_DROME Drosophila hedgehog protein (SWISS-PROTQ02936); SONHHmou, routine sonic hedgehog protein (GenPept X76290); SONHH Brer, Brachyodaniorerio (zebrafish) sonic hedgehog protein (GenPeptZ35669); HH, Xenla, Xenopuslaevis hedgehogprotein (GenPeptL35248). Known intein¢ontaining proteins: PpsZ Mlepr, functionally uncharacterized,intein-containingMycobacteriumleprae protein (GenPept U00013_9); VATA_CANTR,Candida tropicalis vacuolar ATPase subunit (SWISS-PROT P38078); VATA_YEAST,Saccharomyces cerevisiae vacuolar ATPase subunit (SWISS-PROTP17255); POL Pyrsp, Pyrococcussp. DNA polymerase (GenPeptD29671); RECA_MYCLEMycobacteriumleprae RecA ATPase (SWISS-PROT P35901); RECA_MYCTU, Mycobacterium tuberculosis RecA ATPase (SWISS~ROT P26345). The distances from the protein termini are indicated by numbers. Asterisks show identical residues and colons show similar residues between the XenopusHH protein and Ppsl. Slashes show the (putative) cleavage site. The exclamation mark indicatesthe predicted catalytic cysteine. Consensus I shows the sequence conservation between the hedgehogproteins and the more closely related intein-extein junctions in Ppsl and vacuolar ATPase;consensus 2 shows the conservation in all aligned sequences. Upper-case letters indicate residues that are conserved in all sequences in the given group and lower-case letters indicate residues conserved in the majority of sequences. The residues conforming to the consensus are highlighted in bold. U(u) indicates a bulky hydrophobic residue (I, L, V, M, F, Y or W); n indicates a nonpolar residue (I. L, V, M, F, Y, W, A or C); o indicates a small residue (G, A or S); and dot indicates any residue.
141
PROT ..........
UNCE
......... ~'lll......... r...............
MOTMFS
Conversely, when the aligned HH sequences were used for NR screening, unique selection of the intein-containing proteins that had cysteine in the N' junction was observed. These findings suggest that the similarity between the protein splice-junction motif and a conserved segment of the HH family proteins (Fig. I) may be functionally rele-~ant. HH, which is thought to be a secreted and/or a membrane protein, acts as a morphogen in conjunction with other proteins encoded by segmentation genes; in particular, it appears to stimulate the expression of wingless and to antagonize the activity of patched 14,1s.A similar morphogen activity has been reported for the vertebrate homologs of HH~6,~. Evidence of specific proteolysis of the Drosophila HH protein is available. HH contains an internal signal peptide and has been predicted to be cleaved by signal peptidase between amino acids 84 and 85 (Ref. 18). When the mRNA encoding HH was translated in a cell-free system in the presence of microsomes, such a cleavage was indeed apparent, albeit at a relatively low efficiency~s-z°.In addition to the complete protein and the protein presumably produced by the signal cleavage, at least two smaller products wlth molecular masses of 19 kDa and 25 kDa have been observed Is-2°. These values are in very good agreement with the size of putative products of HH cleavage at the predicted site (Fig. I). After removal of the signal peptlde, a single cleavage upstream of the conserved cystelne (Fig. I) would result in an amlno-terminal fragment containing 172 amino acid resldues (19.5 kDa) and a carboxy-termlnal fragment of 216 residues (23.8 kDa). The presence of apparent additional cleavage products IT49raises the possibility of more than one cleavage event, and perhaps even splicing, during HH processing. However, the HH sequences did not show convincing similarity to the C' splice-junction motif of the Inteln-containlng proteins and, therefore, it was not possible to predict the second cleavage site. A series of experiments published very recently, and after the original version of this manuscript had been submitted, has convincingly shown that the HH cleavage Is autocatalytic~0,u. It has also been shown that autoproteolysls is essential for developmental signaling by HH, even though it remains unclear whether different cleavage products have distinct signaling activities. The authors of the study demonstrating the autocatalytic proteolysls of HH2°have pointed out a conserved sequence in the HH family proteins that showed some resemblance to the motif surrounding the catalytic histidine of serine proteases. However, this assignment could not be confirmed
142
TIBS 2 0 - APRIL 1 9 9 5
statistically and its significance remains uncertain. Site-directed mutagenesis of the splicejunction cysteine has invariably abolished splicing in intein-containing proteins 22'23. On t~e other hand, very few, if any, amino acid residues uPstream of the N' junction were required for splicing 23.Similarly, removal of residues 89-254 in the Drosophila HH protein, which left intact only two residues upstream of the proposed cleavage site (Fig. 1), did not impair autoproteolysis 2°. Combining these observations with the results of sequence analysis, I predict that the cysteine that is conserved in HH and in extein-intein splice junctions in both cases acts as a nucleophile in peptide-bond hydrolysis~4. Further, detailed analysis of the HH proteolysis products will show whether these important developmental proteins undergo splicing or aatoproteolysis only.
Acknowledgements ! am grateful to S. M. Dracheva, D. Landsman and S. Pietrokovski Iur stimulating discussions, and to S. Pietrokovsld for sharing his results before publication.
4 Neff, N. E (1993) Curt. Opin. Cell Biol. 5, 971-976 5 Pieffokovski,S. (1995) Protein Sci. 3, 2340-2350 6 Perler,E B. et aL (1994) Nucleic Acids Res. 22, 1125-1127 7 Perler, F. B. et al. (1992) Proc. Nati Acad. Sci. USA 89, 5577-5581 8 Davis, E. O. et aL (1992) Cell 71, 201-210 9 Xu, M. Q. et aL (1993) Cell 75,1371-1377 10 Doolittle,R. F. (1993) Proc. Nail Acad. Sci. USA 90, 5379-5381 11 Hodges,R. A. et aL (1992) Nucleic Acids Res. 20, 6153-6157 12 Altschul, S. F. et al. (1990) J. MoL Biol. 215, 403-410 13 Tatusov, R. L., Altschul, S. F. and Koonin, E. V. (1994) Proc. Nail Acad. ScL USA 91, 12091-12095 14 NOsslein-Volhard,C. and Wieschaus, E. (1980) Nature 187,795=80J 15 Heemskerk,J. and DiNardo, 8. (1994) Cell 76, 449-460 16 Echelard,Y. et al. (1993) Cell 75, 1417-1430 17 Roelink, H. et al. (1994) Cell 76, 761-775 18 Lee, J. J. et aL (1992) Cell 71, 33-50 19 Tabata,T. and Kornberg,1". B. (1994) Cell 76, 89-102 20 Lee, J. J. (1994) Science 266, 1528-1537 21 Peifer, M. (1994) Science 266, 1492-:H93 22 Hirata, R. and Anraku,Y. (1992) Biochem. Biophys. Res. Commun. 188, 40-47 23 Cooper,A. A. et al. (1993) EMBOJ. 12, 2575-2583 24 Wallace,C. J. (1993) Protein ScL 2, 697-705
References 1 Hirata, R. et al. (1990) J. Biol. Chem. 265, 6726-6733 2 Kane,P. M. et al. (1990) Science 250, 651-657 3 Shub, D. A. and Goodrich-Blair,H. (1992) Cell 71, 183-186
EUGENE V. KOOI~IN National Centerfor Biotechnologyinformation, National Libraryof Medicine, National Institutesof Health, Bethesda, MD 20894, USA.
TIBS newsgreup TIBS now has an electronic discussion group called
T l ~ / b l e n e t Joumals.lettem.t|bs. The newsgroup will act as an electronic 'Letters to the Editor', and as a forum for discussion of topcal issues in biochemistry and molecular biology. To send a contribution to the newsgroup, email your message to: [email protected] if you are located in Europe, Africa or central Asia, or tibs@net,bio,net if you are located in the Americas or the Pacific Rim. Please specify the theme of your message clearly in the 'subject' heading and include a brief introduction in the body of your posting (for example, mention the TIBS article to which you are referring). This will allow the discussion to be followed more easily on the newsgroup. All contributions are reviewed by Dr Johanna McEntyre, the TIBS Staff Editor, and Dr David Kristofferson, the BIOSCl newsgroups manager, to act as a filter for messages that might be construed as libellous or that contain purely personal attacks. Contributions will be considered for publication in future issues of TIBS. To receive postings to the group by email, send the command as the main message to the address [email protected];uk if in Europe, Africa or Central Asia, or subs©dbe tibs to the address blesei4erveNJnet.bio.Mt if you are located in the Americas or the Pacific Rim.
SUBbionet-news.ble,et,Jeamals.lettem.tibs
The best way to submit items to the TIBS newsgroup is to use USENET software to post your contribution directly into bionet.Joumais.lettem.tlbs using you~ local newsreading software. You may have to consult your local computer support person to get started, but this small investment of time up front will save you much more time later. News software will sort discussior, s by topic to facilitate brewsing of contributions, and stop your personal email from getting cluttered with messages.
T~BS 2 0 - APRIL 1 9 9 5 11 E~en, ~. L L. et aL (1993) Gone132, 143-148 ./.2 Klenk, H-P.and Doo~itt~e,W. E {1994) Curt. BioL 4, 920-922 13 OuzounisC. and Sander, C. (1992) Ce~ 71, 189-199 i 4 Marsh, T. L., Reich, C. L, White~ock,R. B.and OIsen, G. J. {1994) Prec. Nat/Acad. ScL USA 9!, 4180-4.!84 15 Row~ands,T., Baumann, P. and Jackson, S. P. (1994) Science264,1326=1329
Protein splicing is a recently discovered, complex post-translational modification that includes two concerted proteolytic cleavages and one ligaiion reacUon, and produces two distinct, functional proteins from a single polypeptide ~-s. By analog,:, with introns and exons in RNA precursors, the internal protein that is excised from the translation product is called intein, whereas the two terminal portions, when ligated, form a fusion protein called extein ~. So far, the intein-extein organization has been observed in about ten proteins from yeast, bacteria and Archaea; in most of these cases, the inteins are inserted within or near nucleotide-binding domains ~,5.Protein splicing is thought to be an autocatalytic process, since it can occur in heterologous systems; for example, intein-contalning proteins from Mycobacterium and Thermococcus have been synthesized in Escherichia coilTM, and protein splicing has been demonstrated with the purified inteincontaining DNA polymerase from
~tOCOCCUS9. Comparative analysis of the amino acid sequences of spliced proteins has revealed several conserved motifs (Ref. 5 and references therein). With only one exception, all the inteins are related to the yeast homing endonuclease (HO) and contain two dodecapeptide motifs that are typical of members of a large superfamily of endonucleases, mostly encoded by mobile group I introns ~,m. However, the endonuclease activity is not required for protein splicino . Two other conserved motifs are found around the intein--extein splice junctions ~,s.The amino-terminal (N~)junction motif is found in those of the inteins that have cysteine at the splice site, while inteins that have serine in this position show very little, if any, sequence conservation. The carboxy-terminal (C') junction motif is conserved in all known inteins and contains invariant histidine and asparagine residues 4,s. Here, I describe a
16 Langer, O.and ZiHig, W. (1993) Nucleic Adds Res. 21, 2251-2251 17 Kaine, ~. P., Mehr, O.J. and Woese, C. R. (1994) Prec. Natl Acad. ScL USA 91, 3854-3856 ~80uzounis, C. A., Kyrpides, N. C. and Sander,C. Nucleic Acids Res. {in press) ~90uzounis, C. A. and ~yrpides, N. C. (1994) J. Mol. Evol. 39, 101-104 20 Woese,C. R. (1987) MicmbieLRev. 51, 221-271
Eurcpean Molecular 8iolo~ ~ ~:,:~ory, Postfach 102209, D-69102 Hei~lberg, Germany (current address: A~Center, SR~~nternat~onaL 333 RavenswoodAve,M~nloPa~, CA 94025-34~o3. USA).
protein sptice-~unction motif in the hedgehog (lift) family of eukaryot~c developmental regulatory proteins, and discuss the possibility that it is involved in autoproteolysis and perhaps splicing of these proteins. When the amino acid sequences of the known intein-containing proteins were compared with a nonredundant database @~R)using the BLASTP program ~, moderate similarity (score of 76, probability of occurring by chance approximately 0.08) was observed between the N' splice junction motif of Mycobacterium Ppsl protein s and a segment of HH, a developmental regulatory protein from Xenopus laevis.
Somewhat lower scores were observed between the splice-~undion motifs from Ppsl and yeast vacuolar ATPases, and HH-related proteins from Drosophila to mammals. The region of similarity included the junction motif, and the splice-site cysteine was conserved in all the sequences (Fig. 1). The block of aligned cysteine-contalnlng splice junctions was converted into a positiondependent weight matrb~, which was used to search the NR iteratively ~. The HH family proteins were the only sequences selected; the probability of the HH sequences matching the motif by chance, given the size of the database, was computed to be between 10-4 and 10~.
institute of ~olecu~ar 8iolog~~nd ~iotechnelogyo PO ~o~ 1527, GR-71110oHerak~ior~,Gree~.
| s.on...G/CU ..... u.U..go...U..U..C,d.~ consensus 1 HH_DRO~ 249 81SSHVHG/CFTPESTALLE°SGVRKPLGELSIgDRVL 186 SONHH mou 190 SLAVRAGG/CrPGNATVRLR-SGEP~GL~LHRGD~A~L 173 SOR~H ~ e ~ 185 S%~SGG/CrPGSALVSLO-DGGQ~AVKDT~PGDKVL 193 219 HH Xenla 190 S V A ~ S G G / C F P A G A R V M V E - ~ G T K A V K D ~ R P ~ D R V L Ppsl Mlep~ 193 NTA%~SGG/CLTADARZ~-G~GLVSZADVQP~DEV~ ~ATA_CA~TR 275 S D V X ~ Y V ~ / C ~ T ~ G T ~ - D ~ A D K S Z E S Z E V G D K ~ VATA~YEAST 275 $ D A Z ~ G / C ~ A K G T ~ - D ~ S ~ R C Z E N Z E V G ~ K ~
640 778 761
Pyrsp 398 N I V Y L D F I / C H P A D T ~ - G K G I I N Z S E V O E G D Y V L
135
POL
R E C A ~ Y C L E 198 E E Z G V J ~ G / ~ S T R V T ~ - D G S T E K Z G K Z V N N E M D V REC~CTU ~43 R ~ N K / C L A E G T R Z F D P ~ I T G T T H R I E D V V D G R ~ P X . . . n . . . g / ~ ..... u.u..go...u..U..gd.uU consensus 2
473 510
Rgure1 The N' splice-junction motif in the hedgehogfamily proteins and in intein¢ontaining proteins. The alignment was generated by a combination of CAP and MoST programs~, and a gap was introduced to achieve the optimal alignment of the RECA_MYCTUsequence. The protein sequences were from SWISS-PROTor from GenPept if a SWISS-PROTentry was unavailable. Hedgehogfamily proteins: HH_DROME Drosophila hedgehog protein (SWISS-PROTQ02936); SONHHmou, routine sonic hedgehog protein (GenPept X76290); SONHH Brer, Brachyodaniorerio (zebrafish) sonic hedgehog protein (GenPeptZ35669); HH, Xenla, Xenopuslaevis hedgehogprotein (GenPeptL35248). Known intein¢ontaining proteins: PpsZ Mlepr, functionally uncharacterized,intein-containingMycobacteriumleprae protein (GenPept U00013_9); VATA_CANTR,Candida tropicalis vacuolar ATPase subunit (SWISS-PROT P38078); VATA_YEAST,Saccharomyces cerevisiae vacuolar ATPase subunit (SWISS-PROTP17255); POL Pyrsp, Pyrococcussp. DNA polymerase (GenPeptD29671); RECA_MYCLEMycobacteriumleprae RecA ATPase (SWISS-PROT P35901); RECA_MYCTU, Mycobacterium tuberculosis RecA ATPase (SWISS~ROT P26345). The distances from the protein termini are indicated by numbers. Asterisks show identical residues and colons show similar residues between the XenopusHH protein and Ppsl. Slashes show the (putative) cleavage site. The exclamation mark indicatesthe predicted catalytic cysteine. Consensus I shows the sequence conservation between the hedgehogproteins and the more closely related intein-extein junctions in Ppsl and vacuolar ATPase;consensus 2 shows the conservation in all aligned sequences. Upper-case letters indicate residues that are conserved in all sequences in the given group and lower-case letters indicate residues conserved in the majority of sequences. The residues conforming to the consensus are highlighted in bold. U(u) indicates a bulky hydrophobic residue (I, L, V, M, F, Y or W); n indicates a nonpolar residue (I. L, V, M, F, Y, W, A or C); o indicates a small residue (G, A or S); and dot indicates any residue.
141
PROT ..........
UNCE
......... ~'lll......... r...............
MOTMFS
Conversely, when the aligned HH sequences were used for NR screening, unique selection of the intein-containing proteins that had cysteine in the N' junction was observed. These findings suggest that the similarity between the protein splice-junction motif and a conserved segment of the HH family proteins (Fig. I) may be functionally rele-~ant. HH, which is thought to be a secreted and/or a membrane protein, acts as a morphogen in conjunction with other proteins encoded by segmentation genes; in particular, it appears to stimulate the expression of wingless and to antagonize the activity of patched 14,1s.A similar morphogen activity has been reported for the vertebrate homologs of HH~6,~. Evidence of specific proteolysis of the Drosophila HH protein is available. HH contains an internal signal peptide and has been predicted to be cleaved by signal peptidase between amino acids 84 and 85 (Ref. 18). When the mRNA encoding HH was translated in a cell-free system in the presence of microsomes, such a cleavage was indeed apparent, albeit at a relatively low efficiency~s-z°.In addition to the complete protein and the protein presumably produced by the signal cleavage, at least two smaller products wlth molecular masses of 19 kDa and 25 kDa have been observed Is-2°. These values are in very good agreement with the size of putative products of HH cleavage at the predicted site (Fig. I). After removal of the signal peptlde, a single cleavage upstream of the conserved cystelne (Fig. I) would result in an amlno-terminal fragment containing 172 amino acid resldues (19.5 kDa) and a carboxy-termlnal fragment of 216 residues (23.8 kDa). The presence of apparent additional cleavage products IT49raises the possibility of more than one cleavage event, and perhaps even splicing, during HH processing. However, the HH sequences did not show convincing similarity to the C' splice-junction motif of the Inteln-containlng proteins and, therefore, it was not possible to predict the second cleavage site. A series of experiments published very recently, and after the original version of this manuscript had been submitted, has convincingly shown that the HH cleavage Is autocatalytic~0,u. It has also been shown that autoproteolysls is essential for developmental signaling by HH, even though it remains unclear whether different cleavage products have distinct signaling activities. The authors of the study demonstrating the autocatalytic proteolysls of HH2°have pointed out a conserved sequence in the HH family proteins that showed some resemblance to the motif surrounding the catalytic histidine of serine proteases. However, this assignment could not be confirmed
142
TIBS 2 0 - APRIL 1 9 9 5
statistically and its significance remains uncertain. Site-directed mutagenesis of the splicejunction cysteine has invariably abolished splicing in intein-containing proteins 22'23. On t~e other hand, very few, if any, amino acid residues uPstream of the N' junction were required for splicing 23.Similarly, removal of residues 89-254 in the Drosophila HH protein, which left intact only two residues upstream of the proposed cleavage site (Fig. 1), did not impair autoproteolysis 2°. Combining these observations with the results of sequence analysis, I predict that the cysteine that is conserved in HH and in extein-intein splice junctions in both cases acts as a nucleophile in peptide-bond hydrolysis~4. Further, detailed analysis of the HH proteolysis products will show whether these important developmental proteins undergo splicing or aatoproteolysis only.
Acknowledgements ! am grateful to S. M. Dracheva, D. Landsman and S. Pietrokovski Iur stimulating discussions, and to S. Pietrokovsld for sharing his results before publication.
4 Neff, N. E (1993) Curt. Opin. Cell Biol. 5, 971-976 5 Pieffokovski,S. (1995) Protein Sci. 3, 2340-2350 6 Perler,E B. et aL (1994) Nucleic Acids Res. 22, 1125-1127 7 Perler, F. B. et al. (1992) Proc. Nati Acad. Sci. USA 89, 5577-5581 8 Davis, E. O. et aL (1992) Cell 71, 201-210 9 Xu, M. Q. et aL (1993) Cell 75,1371-1377 10 Doolittle,R. F. (1993) Proc. Nail Acad. Sci. USA 90, 5379-5381 11 Hodges,R. A. et aL (1992) Nucleic Acids Res. 20, 6153-6157 12 Altschul, S. F. et al. (1990) J. MoL Biol. 215, 403-410 13 Tatusov, R. L., Altschul, S. F. and Koonin, E. V. (1994) Proc. Nail Acad. ScL USA 91, 12091-12095 14 NOsslein-Volhard,C. and Wieschaus, E. (1980) Nature 187,795=80J 15 Heemskerk,J. and DiNardo, 8. (1994) Cell 76, 449-460 16 Echelard,Y. et al. (1993) Cell 75, 1417-1430 17 Roelink, H. et al. (1994) Cell 76, 761-775 18 Lee, J. J. et aL (1992) Cell 71, 33-50 19 Tabata,T. and Kornberg,1". B. (1994) Cell 76, 89-102 20 Lee, J. J. (1994) Science 266, 1528-1537 21 Peifer, M. (1994) Science 266, 1492-:H93 22 Hirata, R. and Anraku,Y. (1992) Biochem. Biophys. Res. Commun. 188, 40-47 23 Cooper,A. A. et al. (1993) EMBOJ. 12, 2575-2583 24 Wallace,C. J. (1993) Protein ScL 2, 697-705
References 1 Hirata, R. et al. (1990) J. Biol. Chem. 265, 6726-6733 2 Kane,P. M. et al. (1990) Science 250, 651-657 3 Shub, D. A. and Goodrich-Blair,H. (1992) Cell 71, 183-186
EUGENE V. KOOI~IN National Centerfor Biotechnologyinformation, National Libraryof Medicine, National Institutesof Health, Bethesda, MD 20894, USA.
TIBS newsgreup TIBS now has an electronic discussion group called
T l ~ / b l e n e t Joumals.lettem.t|bs. The newsgroup will act as an electronic 'Letters to the Editor', and as a forum for discussion of topcal issues in biochemistry and molecular biology. To send a contribution to the newsgroup, email your message to: [email protected] if you are located in Europe, Africa or central Asia, or tibs@net,bio,net if you are located in the Americas or the Pacific Rim. Please specify the theme of your message clearly in the 'subject' heading and include a brief introduction in the body of your posting (for example, mention the TIBS article to which you are referring). This will allow the discussion to be followed more easily on the newsgroup. All contributions are reviewed by Dr Johanna McEntyre, the TIBS Staff Editor, and Dr David Kristofferson, the BIOSCl newsgroups manager, to act as a filter for messages that might be construed as libellous or that contain purely personal attacks. Contributions will be considered for publication in future issues of TIBS. To receive postings to the group by email, send the command as the main message to the address [email protected];uk if in Europe, Africa or Central Asia, or subs©dbe tibs to the address blesei4erveNJnet.bio.Mt if you are located in the Americas or the Pacific Rim.
SUBbionet-news.ble,et,Jeamals.lettem.tibs
The best way to submit items to the TIBS newsgroup is to use USENET software to post your contribution directly into bionet.Joumais.lettem.tlbs using you~ local newsreading software. You may have to consult your local computer support person to get started, but this small investment of time up front will save you much more time later. News software will sort discussior, s by topic to facilitate brewsing of contributions, and stop your personal email from getting cluttered with messages.