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Genomics-Proteomics-Bioinformatics(2003).

Genomics-Proteomics-Bioinformatics(2003).

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Letter
Molecular Phylogenetics and Functional Evolution of Major RNARecognition Domains of Recently Cloned and Characterized Au-toimmune RNA-Binding Particle
Erhan S¨uleymanoˇglu
Medical Faculty, Vienna Biocenter, Institute of Biochemistry, University of Vienna, Vienna, Austria.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are spliceosomal macromole-cular assemblages and thus actively participate in pre-mRNA metabolism. Theyare composed of evolutionarily conserved and tandemly repeated motifs, whereboth RNA-binding and protein-protein recognition occur to achieve cellular activ-ities. By yet unknown mechanisms, these ribonucleoprotein (RNP) particles aretargeted by autoantibodies and hence play significant role in a variety of humansystemic autoimmune diseases. This feature makes them important prognosticmarkers in terms of molecular epidemiology and pathogenesis of autoimmunity.Since RNP domain is one of the most conserved and widespread scaffolds, evo-lutionary analyses of these RNA-binding domains can provide further clues ondisease-specific epitope formation. The study presented herein represents a se-quence comparison of RNA-recognition regions of recently cloned and character-ized human hnRNP A3 with those of other relevant hnRNP A/B-type proteins.Their implications in human autoimmunity are particularly emphasized.Key words: hnRNP proteins, RNA folding, RNA-protein interactions, molecular evolution
Introduction
The enclosure of eukaryotic genomes within the nu-clear envelope evolutionarily generated the necessityto transport macromolecules selectively between nu-cleus and cytoplasm. Following their synthesis in cy-toplasm, histones and nucleic acid polymerases haveto reach the nucleus, while specifically cytoplasmicproteins have to be kept out. Mature mRNA, tRNAand rRNA molecules and their associated proteinshave to follow the opposite route, while their imma-ture precursors have to be kept in (
1–5 
). Eukary-otic mRNA is enzymatically metabolized and com-pacted with proteins within nuclei to generate func-tional messenger ribonucleoprotein (mRNP) particles(
,
). The control of protein biosynthesis involvesregulation of intranuclear functions with participationof specific proteins, many of which seem to enter fromthe cytoplasm as a time-specific event (
3–7 
). Theheterogeneous nuclear ribonucleoproteins (hnRNPs)comprise a group of important regulators engaged inthese cellular processes (
8–10 
).Extensive research efforts have been devoted tothe cloning and molecular characterization of numer-ous ribonucleoproteins (RNPs) in the context of their
E-mail: erhan@e-mail.dk;erhan@mail-online.dk
active role in mRNA biogenesis and metabolism (
1,2, 9–11
), as well as in terms of their involvement asautoantigens in human diseases (
11
). RNA-proteininteractions are considered as primary macromolecu-lar forces governing gene expression. Hence, emphasisis put on the roles of various RNA-binding motifs andtheir subsequent roles in governing cellular activities,both in health and pathology (
). Recently, therehas been considerable interest in the participation of these RNA-binding particles in triggering the immuneresponse in human autoimmune disorders (
11
).Substantial research both at cDNA and proteinlevel have been performed on immunochemical fea-tures of human autoantigens (http://www.zoo.uni-heidelberg.de/mol evol/MB/ana base.html). Of par-ticular interest is the group of mammalian hnRNPs,which constitutes a part of spliceosome, which itself is an autoimmune target (
12 
). Novel nucleotide se-quences are continuously reported, which afterwardsare used to delineate homology among various mem-bers based on previously determined nucleotides andprotein sequences.hnRNPs of diverse origin possess a common well-known structural motif. However, the functionalchemistry of these domains remains unknown. Themechanism of contribution of numerous hnRNPs witha similar structural RNP motif (
13 
) to the induc-310 Geno., Prot. & Bioinfo. Vol. 1 No. 4 November 2003
 
uleymanoˇglu
tion of different immune reactions is sequence-specific(
14
). Phylogenetic approach is essential to find func-tionally important genomic sequences based on detec-tion of their high degree of conservation across differ-ent species. Such approach shows the level of improve-ment of the prediction of gene-regulatory elements inthe human genome. This necessitates the study of thedegree of homology of RNA recognition motifs (RRM)among these proteins, requiring an evolutionary com-putation. Having considered the importance of sub-mitting new sequences for further functional charac-terization, the newly cloned and expressed cDNA of previously unknown member of the hnRNP A/B fam-ily of proteins (Figure 1) is presented here. Its RNA-binding properties and tissue-specific gene expressionprofiles were recently determined (
15 
). Based on theconcept of correlation between sequences and RNA-binding modes, a systemic search was performed fornucleic acid association by evaluating sequence con-servation using multiple sequence alignments searchtools. This study continues phylogenetic results ob-tained from previous larger data sets (
16 
).
Fig. 1
The general 2xRNA-binding domain (RBD)—glycine structure of hnRNP A3. The space between thetwo adjacent RBDs is occupied by inter-RNA recognitionmotif linker fragment (IRL). Amino acids 1–209 compriseboth RBD1 and RBD2. RBD1 alone is composed of frag-ments of amino acids 1–112, while RBD2 is from aminoacids located in positions 112–209. Glycine-rich domaincontains amino acids numbered 209–296.
Results
The aim of the presented work herein was to iso-late novel cDNA sequences with important functionalimplications in human pathology. Our efforts havebeen devoted to the cloning and subsequent tissue-specific gene expressions of numerous human RNPsfrom the hnRNP A/B family of proteins (Figure 1;ref.
15 
). The objective was to search for molecularbasis of autoimmunity by applying comparative anal-ysis of the sequences of diverse autoantigens. In thiscontext, evolutionary computation approach couldgive us major clues on how evolutionarily conservedmRNA transport machinery fails are linked to devel-opment of human autoimmune disorders. We weremainly interested in cDNAs, which might encode theyet undescribed hnRNP B2. The need for this wasbased on two observations. On the one hand, autoan-tibodies directed against hnRNP A2 crossreact withhnRNP B1 and hnRNP B2. Since hnRNP B1 is an al-ternatively spliced variant of hnRNP A2, this suggeststhat hnRNP B2 might be an alternatively spliced formof hnRNP A2/B1. However, no attempts to clone acDNA encoding hnRNP B2 were successful so far. Onthe other hand, cDNAs closely related to hnRNP A1and hnRNP A2 have been previously isolated from ahuman fetal brain library and from a
Xenopus laevis
library, respectively. Their close relationships withhnRNP A2 suggested that one of these cDNAs mightactually encode hnRNP B2 (
15 
).To isolate the searched cDNA, human liver andbrain cDNA expression libraries were screened byPCR using primers complementary to 5
- and 3
-untranslated regions of the FBRNP cDNA. The iso-lated sequence seemed to encode the full-length pro-tein. Interestingly, however, it was not completely ho-mologous to the FBRNP cDNA. Since the obtainednew sequence shared close identity to the
Xenopuslaevis
hnRNP A3 cDNA sequence (Entrez; accessionnumber L02956), the protein was termed human hn-RNP A3.Nucleotide sequence comparisons betweenFBRNP,
Xenopus laevis
hnRNP A3 and our newlydetermined human hnRNP A3 proteins revealed thatextensive sequence conservation exist in RNA-bindingregions. The differences observed here were mainly atthe third position of the codon triplet. The majorityof sequence variations were seen at the Gly-rich do-main, composed of amino acids at positions 211-373.These sequences were observed more at nucleotidelevel, as expected, compared to the translated pro-tein sequences (Figure 2). Only protein sequences areshown for brevity.Identification of various nucleic acid-binding do-mains of diverse hnRNPs was achieved by cloningand sequencing of cDNAs encoding these motifs. Ingeneral, all known human hnRNP proteins containat least one RNA-binding module and one anotherauxiliary domain fragment. The RNA-binding motifscontain the RNP consensus sequences (CS-RBD), theRNA recognition motif (RRM; ref.
10 
,
13 
,
17 
), theRNP-80 motif, the RGG box (
18 
), and the KH do-main (
19 
). RNP domain is the most common featurein these RNPs. This domain is found in hnRNPs invarious amounts, ranging from 1 (in hnRNP C) to 4(
e. g.
in Poly A-binding protein; ref.
10 
). Figure 1shows the general modular structure of hnRNP A/Btype of RNP particles. Their general structure is com-posed of two domains: the first 195 residues comprisethe so-called UP1 domain, containing two canonicalRNA-recognition motifs (RRM 1 and RRM 2), eachof which is comprised from the conserved RNP-2 andRNP-1 submotifs. The Gly-rich C-terminal domainGeno., Prot. & Bioinfo. Vol. 1 No. 4 November 2003 311
 
hnRNP A/B Proteins
Fig. 2
Deduced protein sequences of 
Xenopus laevis
hnRNP A3, FBRNP, and human hnRNP A3. The sequencesare aligned and displayed using the CLUSTALW programme, as mentioned in Materials and Methods. The alignmentspans 373 amino acid residues. Dashes (–) represent apparent deletions or insertions; colons (:) mark semi-conservativeamino acid exchanges; periods (.) depict more distantly located residues; and asterisks (*) show conserved residues.Amino acids of FBRNP and
Xenopus laevis
hnRNP A3, respectively, differing from human hnRNP A3, are shown inbold type.
comprises an RGG box and a nuclear localization mo-tif. This motif contributes to protein-protein interac-tion patterns, as well as to subcellular localization(
18 
). Two conserved solvents exposed Phe residuesat the centre of the
β 
-sheet in each RRM-contactedRNA. The least conserved 3
loop in U1A is engagedin extensive RNA interactions, whose conformationchanges upon RNA binding (
17 
,
18 
,
20 
). The RNPdomain interacts with a flexible single-strand RNAand the
β 
-sheet provides a large surface for extensiveinteraction with nucleotides. Regions outside of theRRM may also play important roles in RNA binding(
10 
). Identification of these motifs as RNA-bindingdomains has been used for prediction of this activityin various proteins of yet undescribed function pos-sessing these domains.As seen in Figure 2, the newly isolated human hn-RNP A3 cDNA encodes a protein of 296 amino acids(a.a.). The calculated molecular weight of 32 kDawas also confirmed electrophoretically. Amino acidsnumbered 1–98 comprise RBD1, 112–209 compriseRBD2, 209–296 comprise the Gly-rich domain, while99–111 contain inter-RRM linker (IRL) segments. Toour surprise, comparison with the previously reportedFBRNP cDNA sequence, encoding a 269 a.a. pro-tein, revealed only 85% identity. Therefore, it was as-sumed that the currently presented cDNA encodes anovel and yet undescribed protein. Despite the closehomologies among the RNA-binding regions (95%),remarkable differences can be seen in the C-terminaldomain (Figure 2), where deletions and insertions areapparent. Thus, the FBRNP is 27 a.a. shorter andthere are fewer conserved residues in the C-terminalpart. Both cDNAs also show high homologies to thecDNA encoding hnRNP A3 from
Xenopus laevis
ex-cept for a stretch of 14 a.a. at the RBD1 N-terminalpart. The RNA-binding regions of the three proteinsare almost identical, while the auxiliary domains areless conserved. The
Xenopus laevis
protein shows75% homology with FBRNP and 83% with the newlycloned human hnRNP A3 protein, respectively (Fig-ure 2). The reduced length of the human hnRNP A3,as compared to the
Xenopus laevis
homologue (296vs. 373 a.a.), is compatible with differences observedbetween
Xenopus laevis
and rat hnRNP A1, which arecomposed of 365 and 320 residues, respectively (
15 
).312 Geno., Prot. & Bioinfo. Vol. 1 No. 4 November 2003

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