Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC 50 ≥ 6.5 nM) and high affinity ligands ( K D ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.

Ullu, 1990) and occur in the form of snRNPs in the cell S.Lücke, T.Klöckner and Z.Palfi contributed equally to this work (Michaeli et al., 1990;Cross et al., 1991;Günzl et al., 1992). The SL RNP is an additional, trans-splicing specific In trypanosomes all mRNAs are generated through factor and has been proposed as a U1 snRNP equivalent trans mRNA splicing, requiring the functions of the (Bruzik et al., 1988; discussed in Steitz, 1992); although small nuclear RNAs U2, U4 and U6. In the absence of it contributes its miniexon during the trans-splicing reacconventional cis mRNA splicing, the structure and tion, it also resembles an snRNP on the basis of its protein function of a U5-analogous snRNP in trypanosomes composition . Using affinity purification has remained an open question. In cis splicing, a U5of Trypanosoma brucei snRNPs, five common lowsnRNP-specific protein component called PRP8 in yeast molecular-weight polypeptides have been identified that and p220 in man is a highly conserved, essential splicing are shared by the U2, U4/U6 and SL RNPs; in addition, factor involved in splice-site recognition and selection.this analysis revealed several snRNP-specific protein comWe have cloned and sequenced a genomic region ponents . In contrast to the yeast and from Trypanosoma brucei, that contains a PRP8/p220-mammalian cis-splicing systems, only one snRNP protein homologous gene (p277) coding for a 277 kDa protein.gene has been cloned so far from the trypanosome transUsing an antibody against a C-terminal region of splicing system, coding for a U2-specific 40 kDa protein the trypanosomal p277 protein, a small RNA of~65 with homology to the cis-spliceosomal U2 AЈ protein nucleotides could be specifically co-immunoprecipit- . ated that appears to be identical with a U5 RNA (SLA2 An important open question has been whether there is RNA) recently identified by Dungan et al. (1996). Based a trans-spliceosomal homologue of the U5 RNA. U5 RNA on sedimentation, immunoprecipitation and Western is the least conserved among the spliceosomal RNAs, and blot analyses we conclude that this RNA is part of a phylogenetic comparisons showed that essentially only stable ribonucleoprotein (RNP) complex and associated the 11-nucleotide 5Ј loop sequence is conserved (Guthrie not only with the p277 protein, but also with the and Patterson, 1988;Frank et al., 1994). The 5Ј loop of common proteins present in the other trans-spliceo-U5 RNA is functionally important in cis splicing, in somal snRNPs. Together these results demonstrate particular during 5Ј and 3Ј splice-site recognition and that a U5-analogous RNP exists in trypanosomes and selection; this has been established in yeast using in vivo suggest that basic functions of the U5 snRNP are mutational studies Norman, 1991, 1992) conserved between cis and trans splicing.and also in the mammalian splicing system (Wyatt et al., Keywords: snRNA/snRNP/trans splicing/trypanosomes/ 1992; Cortes et al., 1993;Sontheimer and Steitz, 1993

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