the enclosure of eukaryotic genomes within the nuclear envelope evolutionarily generated the necessity to transport macromolecules selectively between nucleus and cytoplasm .
following their synthesis in cytoplasm , histones and nucleic acid polymerases have to reach the nucleus , while specifically cytoplasmic proteins have to be kept out .
mature mrna , trna and rrna molecules and their associated proteins have to follow the opposite route , while their immature precursors have to be kept in 1 . , 2 . , 3 . , 4 . , 5 .. eukaryotic mrna is enzymatically metabolized and compacted with proteins within nuclei to generate functional messenger ribonucleoprotein ( mrnp ) particles 6 .
, 7 .. the control of protein biosynthesis involves regulation of intranuclear functions with participation of specific proteins , many of which seem to enter from the cytoplasm as a time - specific event 3 . , 4 . , 5 .
, 7 .. the heterogeneous nuclear ribonucleoproteins ( hnrnps ) comprise a group of important regulators engaged in these cellular processes 8 . , 9 . , 10 .. extensive research efforts have been devoted to the cloning and molecular characterization of numerous ribonucleoproteins ( rnps ) in the context of their active role in mrna biogenesis and metabolism 1 .
, 2 . , 9 . , 10 . , 11 . , as well as in terms of their involvement as autoantigens in human diseases ( 11 ) .
hence , emphasis is put on the roles of various rna - binding motifs and their subsequent roles in governing cellular activities , both in health and pathology ( 9 ) .
recently , there has been considerable interest in the participation of these rna - binding particles in triggering the immune response in human autoimmune disorders ( 11 ) .
substantial research both at cdna and protein level have been performed on immunochemical features of human autoantigens ( http://www.zoo.uni-heidelberg.de/mol_evol/mb/ana_base.html ) .
of particular interest is the group of mammalian hnrnps , which constitutes a part of spliceosome , which itself is an autoimmune target ( 12 ) .
novel nucleotide sequences are continuously reported , which afterwards are used to delineate homology among various members based on previously determined nucleotides and protein sequences .
the mechanism of contribution of numerous hnrnps with a similar structural rnp motif ( 13 ) to the induction of different immune reactions is sequence - specific ( 14 ) .
phylogenetic approach is essential to find functionally important genomic sequences based on detection of their high degree of conservation across different species .
such approach shows the level of improvement of the prediction of gene - regulatory elements in the human genome .
this necessitates the study of the degree of homology of rna recognition motifs ( rrm ) among these proteins , requiring an evolutionary computation .
having considered the importance of submitting new sequences for further functional characterization , the newly cloned and expressed cdna of previously unknown member of the hnrnp a / b family of proteins ( figure 1 ) is presented here .
its rna - binding properties and tissue - specific gene expression profiles were recently determined ( 15 ) . based on the concept of correlation between sequences and rna - binding modes , a systemic search was performed for nucleic acid association by evaluating sequence conservation using multiple sequence alignments search tools .
the aim of the presented work herein was to isolate novel cdna sequences with important functional implications in human pathology .
our efforts have been devoted to the cloning and subsequent tissue - specific gene expressions of numerous human rnps from the hnrnp a / b family of proteins ( figure 1 ; ref .
the objective was to search for molecular basis of autoimmunity by applying comparative analysis of the sequences of diverse autoantigens . in this context
, evolutionary computation approach could give us major clues on how evolutionarily conserved mrna transport machinery fails are linked to development of human autoimmune disorders .
we were mainly interested in cdnas , which might encode the yet undescribed hnrnp b2 .
the need for this was based on two observations . on the one hand , autoantibodies directed against hnrnp a2 crossreact with hnrnp b1 and hnrnp b2 . since hnrnp b1 is an alternatively spliced variant of hnrnp a2 , this suggests that hnrnp b2 might be an alternatively spliced form of hnrnp a2/b1 .
however , no attempts to clone a cdna encoding hnrnp b2 were successful so far .
on the other hand , cdnas closely related to hnrnp a1 and hnrnp a2 have been previously isolated from a human fetal brain library and from a xenopus laevis library , respectively . their close relationships with hnrnp a2 suggested that one of these cdnas might actually encode hnrnp b2 ( 15 ) .
to isolate the searched cdna , human liver and brain cdna expression libraries were screened by pcr using primers complementary to 5- and 3-untranslated regions of the fbrnp cdna .
since the obtained new sequence shared close identity to the xenopus laevis hnrnp a3 cdna sequence ( entrez ; accession number l02956 ) , the protein was termed human hnrnp a3 .
nucleotide sequence comparisons between fbrnp , xenopus laevis hnrnp a3 and our newly determined human hnrnp a3 proteins revealed that extensive sequence conservation exist in rna - binding regions .
the majority of sequence variations were seen at the gly - rich domain , composed of amino acids at positions 211 - 373 .
these sequences were observed more at nucleotide level , as expected , compared to the translated protein sequences ( figure 2 ) .
only protein sequences are shown for brevity . identification of various nucleic acid - binding domains of diverse hnrnps was achieved by cloning and sequencing of cdnas encoding these motifs . in general , all known human hnrnp proteins contain at least one rna - binding module and one another auxiliary domain fragment .
the rna - binding motifs contain the rnp consensus sequences ( cs - rbd ) , the rna recognition motif ( rrm ; ref .
, 13 . , 17 . ) , the rnp-80 motif , the rgg box ( 18 ) , and the kh domain ( 19 ) .
this domain is found in hnrnps in various amounts , ranging from 1 ( in hnrnp c ) to 4 ( e. g. in poly a - binding protein ; ref .
figure 1 shows the general modular structure of hnrnp a / b type of rnp particles .
their general structure is composed of two domains : the first 195 residues comprise the so - called up1 domain , containing two canonical rna - recognition motifs ( rrm 1 and rrm 2 ) , each of which is comprised from the conserved rnp-2 and rnp-1 submotifs .
the gly - rich c - terminal domain comprises an rgg box and a nuclear localization motif .
this motif contributes to protein - protein interaction patterns , as well as to subcellular localization ( 18 ) .
two conserved solvents exposed phe residues at the centre of the -sheet in each rrm - contacted rna .
the least conserved 3 loop in u1a is engaged in extensive rna interactions , whose conformation changes upon rna binding 17 .
, 20 .. the rnp domain interacts with a flexible single - strand rna and the -sheet provides a large surface for extensive interaction with nucleotides .
regions outside of the rrm may also play important roles in rna binding ( 10 ) .
identification of these motifs as rna - binding domains has been used for prediction of this activity in various proteins of yet undescribed function possessing these domains .
as seen in figure 2 , the newly isolated human hnrnp a3 cdna encodes a protein of 296 amino acids ( a.a . ) .
amino acids numbered 198 comprise rbd1 , 112209 comprise rbd2 , 209296 comprise the gly - rich domain , while 99111 contain inter - rrm linker ( irl ) segments . to our surprise , comparison with the previously reported fbrnp cdna sequence , encoding a 269 a.a .
therefore , it was assumed that the currently presented cdna encodes a novel and yet undescribed protein . despite the close homologies among the rna - binding regions ( 95% ) ,
remarkable differences can be seen in the c - terminal domain ( figure 2 ) , where deletions and insertions are apparent .
shorter and there are fewer conserved residues in the c - terminal part . both cdnas also show high homologies to the cdna encoding hnrnp a3 from xenopus laevis except for a stretch of 14 a.a . at the rbd1 n - terminal part .
the rna - binding regions of the three proteins are almost identical , while the auxiliary domains are less conserved .
the xenopus laevis protein shows 75% homology with fbrnp and 83% with the newly cloned human hnrnp a3 protein , respectively ( figure 2 ) .
the reduced length of the human hnrnp a3 , as compared to the xenopus laevis homologue ( 296 vs. 373 a.a . ) , is compatible with differences observed between xenopus laevis and rat hnrnp a1 , which are composed of 365 and 320 residues , respectively ( 15 ) .
the assumption that the presented clone encodes the human counterpart of frog hnrnp a3 is further reinforced , when conserved residues in the auxiliary domains of the a / b polypeptides are considered .
thus , a stricking homology is apparent at the c - terminus , which is well conserved between frog and human hnrnp a3 with 15 of the last 18 a.a .
both proteins end with a triplet rrf , which is also present in hnrnp a1 , but neither in fbrnp nor in hnrnp a2/b1 . moreover , a glutamic acid located at the boundary of rbd2 and the auxiliary domains of xenopus laevis and human hnrnp a3 ( a.a .
position 207 ) is substituted by valine in fbrnp , a further indication that the novel protein is indeed the human counterpart of xenopus laevis hnrnp a3 .
interestingly , this result is reported also for the murine hnrnp mbx protein , which is highly homologous to both human fbrnp and xenopus laevis hnrnp a3 ( 21 ) .
most of the conformational differences between the two rrms occur either at the c - terminal end of the b helices , or at the tip of loop 3 , which is tilted and twisted in rrm1 , as opposed to rrm2 ( figure 3 ) .
the rnp domain interacts with a flexible single - strand rna and the -sheet provides a large surface for extensive interaction with nucleic acids .
the two aromatic side chains of rnp1 and one aromatic side chain of rnp2 provide a convenient template for base stacking of the rna with neighbouring protein side chains , forming hydrogen bonds with the stacked bases .
although the major groove of the a - type rna helix is too narrow and deep to provide a site for sequence - specific association , an rna loop with exposed nucleotides provides a large surface for protein binding .
however , the rna - binding is not a unique feature of the -sheets . despite the evolutionary conservation of the rrms , which are necessary for both general and sequence - specific nucleic acid binding
, regions outside of the rrms may also play important roles in rna binding ( 10 ) .
for instance , the flexibility of the linker sequence connecting both rrms , resulting from two pairs of arg and asp involved in irl salt bridges and the ordered residues , creates a position which is highly probable for this sequence to be involved in direct rna binding .
figure 3 shows the rrm sequences of 12 hnrnp a / b type of proteins , extending the previous data set of mayeda et al .
there is extensive sequence homology between these various proteins , which are conserved throughout evolution from insects to man .
each of the rrms forms an ungapped alignment with both rrms of hnrnp a1 . in all of the shown proteins ,
the evolutionary links between the rrms of human hnrnp a3 and other hnrnp a / b proteins are depicted in a phylogenetic tree ( figure 4 ) .
the sequences of rrm1 and rrm2 cluster in two separate groupings , representing insect and vertebrate proteins .
the patterns of branching for rrm1 and rrm2 sequences are nearly identical , except for the placement of the branch representing the minor human variant hnrnp a0 .
the almost identical pattern of branching for both rrms suggests that these fragments have evolved in parallel .
obviously , each of the insect and vertebrate proteins is equally distant from each other .
apparently , this fact indicates independent gene duplications of ancestral hnrnp a / b - like protein , in agreement with mayeda et al .
the independent evolution of individual tandemly repeated protein domains and their functional relevance has always being an intriguing issue . the rnp domain is commonly encountered scaffold among the nearly 350 different folds , that is , the favourable secondary arrangements of around 10,000 protein structures covered by the currently used databases 22 . , 23 . , 24 . , 25 .. comparisons of various protein structures , in combination with their nucleotide analyses serve as a clue for their evolution .
the debate is whether these structures have evolved for achievement of specific functions or for thermodynamic stability and/or for kinetic folding reasons .
the same structural topology can determine numerous activities , preserved through evolution . in biological systems , enzymes evolve by acquiring of new thermodynamic or kinetic properties by the already existing protein folds .
generally , only the overall folding pattern is conserved in protein groups , and as the sequence diverges , the structures deform . among the widespread catalytic folds , such as the tim barrel and the globins
, the rrm domain attracts research efforts into delineating the advancements of evolution starting from all  - helical proteins via all -sheet proteins and reaching a group of /-proteins . in this route
, the /-barrel is the most frequently seen enzymatic fold and appears to be a selected topology for the directed evolution of new biocatalysts .
it is worth studying how the present /-barrels are linked evolutionarily to each other and through which way they have evolved from simpler ancestors .
the topic becomes more attractive when these folds comprise a domain involved in a disease state , such as autoimmunity .
, 11 .. the rapidly growing information on structures and functions of numerous hnrnps will be invaluable for understanding the pathological mechanisms by which these particles participate as targets of autoantibodies . of particular interest is the determination of the role of rna - protein folding patterns in development of disease - specific epitope formation . in this context
, the evolution of selected nucleic acid - binding sequences and how this contributes to disease establishment remain unknown .
therefore , this study was devoted to presentation of evolutionary connections between the various hnrnps and their rna - binding domains .
a phylogenetic tree is given to depict these motifs in the major group of these particles , including also the recently cloned and sequenced novel human hnrnp a3 ( 15 ) , followed by remarks on the lastly selected sequences , relevant in diseases . figure 1 shows the general 2xrbd - gly type of structure of the a / b type of human hnrnps .
the two canonical rrm-1 and rrm-2 , containing the conserved rnp-2 and rnp-1 submoieties , and the rgg c - terminal part constitute the main domains .
the translated sequences of the recently characterized human hnrnp a3 particle is presented herein ( figure 2 ) , solely to demonstrate the high degree of sequence homology among the different rrms .
its protein sequence is compared to that of other two members of this protein family 
the selected three members in figure 2 are functionally related quite distantly from each other , each having a buried core tryptophan and there are disulphide bonds on the highly conserved irl - segments ( 16 ) .
the sequence comparisons of the three closely related hnrnp a3 members showed that the general need for a large buried hydrophobic moieties is well satisfied .
this represents a possible case , where structural constraints make evolution towards a different conformation unlikely .
the high level of conservation of these residues is related to the interlocking interactions and restraints of the loop connecting the -sheets , by providing the huge hydrophobic template necessary for core packing .
in addition , having large hydrophobic inner part and suitable hydroxyls capable of hydrogen - bond formation , these residues ( e.g. tyrosines ) could be conserved for structural stability needs . performing amino acid mutations , or substitutions at the sites thus removing hydrophobic packaging interactions , would provide further evidence for the core packing of hnrnp globule .
these are buried in the compacted core and are expected to participate in overall folding of the scaffold .
however , to verify whether this is the case , more mutational analyses , combined with thermodynamic , kinetic , structural and evolutionary computation data are needed .
amino acid sequence comparisons of rrm structures ( figure 3 ) depicted the hydrophobic core residues , conserved among different rrms .
the inserted sequence of the human hnrnp a3 shared extensive sequence homology ( up to 90% ) existing between rrms of all these numerous proteins , which are conserved throughout evolution . a phylogenetic tree ( figure 4 ) depicted the evolutionary history of the rrms of human hnrnp a3 and other hnrnp a / b proteins .
the objective was to deduce whether the observed patterns of conservation resulted due to structural features for this type of fold architecture , or alternatively , were the consequence of divergent evolution .
parallel symmetric groupings of rrm1 and rrm2 in two different clusters , which belong to insect and vertebrate members is well seen . with the slight deviation of hnrnp a0 ,
this branching pattern is an indication of parallel proceeding evolution , in accordance with relevant studies 16 .
, 26 .. each of the members of these two subgroups are equally distant from each other .
these results suggest that these branching patterns originated from an ancestral hnrnp a / b - like protein probably through independent gene duplications .
phylogenetic analyses of this sort provide evidence for evolutionary history and origin of the modular structure of hnrnps and their contemporary functional implications .
this approach helps to delineate whether rna - binding ( rbd ) and protein - protein ( auxiliary ) domains have evolved in parallel or follow another evolutionary history . in this context evolutionary computation on every newly characterized hnrnp particle
the sequence of human hnrnp a3 was inserted in the data set at figure 4 and becomes a further support of the evolutionary trend of these rnp family of proteins , appearing also in previous studies 16 .
, 26 .. these earlier studies proved that the origin of hnrnps is a consequence of independent gene duplication . in their landmark work , fukami - kobayashi et al .
( 26 ) showed that the ancestral gene of the hnrnps had have two rna - binding domains even before the divergence of invertebrates and vertebrates and that it diverged to contemporary hnrnp genes while preserving the tandemly repeated structures .
the origin of natural selection of particular nucleotide sequences and their conservation from primitive rna - based catalysis to eukaryotic chromosome evolution remain to be elucidated ( http://manske.virtualave.net/genetik/vorlesung_ws99/teil1-3/rna_und_die_evolution.htm ; ref .
, there is now sufficient data to prove the substitution of ancestral rnas by more efficient rna - protein complexes with subsequent replacement of the latter by proteins ( 27 ) . by using molecular modelling approach ,
it has been recently shown ( 28 ) that in this transition position , the two rna - binding sites of the simulated proto - protein interact with target sites to form a stable rnp complex . since the selected proteins were unable to resolve misfolded rna , in vivo selected protein associated with the two rna - binding sites , indicating that the protein facilitates the correct folding of the ribozyme .
this provides evidence that these modelled self - splicing rna - protein complexes can be considered as a primitive form of splicing factors or rna chaperones , which contribute to correct rna folding ( 28 ) . in terms of molecular evolution of primitive catalysts ,
these self - splicing intron assemblies possibly became splicing rnps , suggesting that an active rnp particles could have originated from a primordial ribozyme ( 28 ) . the evolutionary driving force for this
is the small structural cost , through which the protein binding compensates for some folding deficiencies in the rna ( 29 ) .
this serves as another proof that the evolution from rna to rnp - determined catalysis represents an evolutionary design against misfolding rather than for the maintenance of a protein binding site .
our preliminary biophysical results ( 30 ) suggest that this is the case with human hnrnp a3 , as well .
the current study presents the sequence conservation of mainly hydrophobic residues of human hnrnp a3 , which form the hydrophobic packing cores and are essentially and evolutionarily conserved in other rna - binding domains in the hnrnps studied ( fig .
this indicates that the rna - binding domains have evolved from a common hnrnp a / b - type proto - protein , supporting further previous models 16 .
, 26 .. interestingly , while the majority of models of rbd protein family built up to now are based on divergent evolutionary principles , another view claims that this is open to doubt in cases of similar , but more distantly related functions and structures ( 31 ) .
their model suggests that even though the ancient rbds could have developed separately by divergent evolution , these rbds have evolved conserved rnp motifs with a similar structure and function on similar surfaces .
the authors emphasize that rnp motifs are conserved both in scaffold architecture and in function , which provides an intriguing case of convergent evolution .
further advancements in sequence comparison techniques will help to understand whether these functional sites have arisen multiple times during evolution ( http://online.itp.ucsb.edu/online/infobio01/higgs1/pdf/higgs1.pdf ; ref .
22 . , 23 . , 24 . , 32 . ) . regarding the evolutionary conservation of hnrnp domains ,
two possibilities exist for explaining their nowadays existence  they are either hyperadaptable , or they may have developed features required to perform vital cellular functions ( 32 ) . despite the case with cold shock domain ( 31 ) , hnrnp a / b proteins appear to possess eukaryotic origins , because prokaryotic homologs could not be detected until now .
all these rbds have similar dimerization roles leading to similar three - dimensional architectures , suggesting that they may have arisen from a common ancestor , which have diverged in sequence afterwards ( 32 ) .
these domains are found also in chloroplast protein sequences , implying the link with endosymbiont hypothesis , and also implys that the multicellularity in plants and animals did not evolved independently 26 .
, 32 .. domain evolution of hnrnps rises the question that whether they are consequences of continuous or discontinuous evolution .
fukami - kobayashi et al . ( 26 ) showed that the rbd fused with the sr - rich domain proceeded the divergence of splicing factors , and that these two domains have arisen together thus conserving the fused domain organization .
the existence of gly - rich domain ( figure 1 ) , also supports the proposal of domain organization of the unique origin of hnrnps , followed by duplication and divergence of rbds and structural transitions of the auxiliary domain ( 18 ) .
this indicates that the auxiliary domain shared by functionally related rnps diverged in parallel together with the rbds ( 26 ) .
it is thus implied , that these ancestral repeats must have oligomerized afterwards to adopt a similar structure seen in contemporary homologs ( 32 ) . in the light of the fact that hnrnp a / b particles
are considered as disease genes , an intriguing issue becomes the use of protein domain data of various hnrnps to delineate the link and etiology of diseases . as stated earlier , the majority of these gene activities related to autoantigenic hnrnps are covered by relevant databases .
respect , identification of human paralogous disease genes generates further pathological gene candidates to be sequenced , because paralogous genes are frequently encountered as mutated versions in similar diseases ( 32 ) .
the sequence comparison , however , may be insufficient for deduction of its functional role for patients diagnozed with the suspected disease .
therefore , we followed the suggestion of ponting , c.s . and russell , r.r .
( 32 ) and combined our sequence determination of human hnrnp a3 with its gene expression profiles , its molecular interaction features ( 30 ) , and its tissue - specific gene expression patterns ( 15 ) .
interestingly , our molecular characterization of human hnrnp a3 showed that while the recombinant hnrnp a3 with its 296 a.a .
migrates as expected as a 32  kda protein on sds - page analysis , it is recognized by the patients sera as a 50  kda protein .
this is attributed to alternative splicing or due to tissue - specific expression of a highly related yet unknown crossreactive protein .
surprisingly , neither the 50  kda nor the 32  kda protein was detected in helanuclear extracts , further supporting the assumption that hnrnp a3 is not ubiquitously expressed as hnrnp a1 or hnrnp a2 .
northern blotting analysis showed the variable expression patterns of hnrnp a3 mrna in human tissues .
thus , it is highly expressed in spleen , ovary , small intestine , lung , liver , skeletal muscle , kidney and pancreas , whereas expression in thymus , testis , colon and peripheral blood was hardly detectable .
expression in prostata , heart , brain and placenta was low , but clearly detectable ( 15 ) .
in addition , circular dichroism and fluorescence spectroscopic measurements demonstrated that the human hnrnp a3 protein is a stable particle  the free energy of unfolding of the full - length hnrnp a3 is 58c , as shown by both urea and temperature denaturation ( 30 ) .
this particle increases its apparent stability upon interaction with rna fragments r(uuaggg)4 to higher temperature values .
the high binding affinity to this repeat indicates its preference for association with purine - rich consensus sequences and targeting features towards deleterious g - tetrad structures .
the latter fact suggests its active participation in alternative splicing  a common feature of well - known telomitic repeats interactions of hnrnp a1 .
this rna fragment , hnrnp a3 particle recognition , can act by facilitating the splicing of alternative intron of the pre - mrna . since this represents a case of regulation of splice - site selection with further functional implication in human disease 11 .
the first one is comprised of both rbd1 and rbd2 , and the second one is composed of rbd2 and certain parts of gly - rich domain 15 . , 30 .. this epitope recognition pattern differs from the epitope determined for the highly related hnrnp a1 and hnrnp a2 , respectively .
thus , in hnrnp 2 the rbd2 was found to contain the major epitope , which was recognized by patients suffering from rheumatoid arthritis or systemic lupus erythenathosus .
this particular region was also found to be essential for interaction with rna and the patients autoantibodies strongly inhibited rna binding . as oppose to these , autoantibodies derived from patients diagnozed with mixed connective tissue disease recognized an epitope comprising rbd1 and rbd2 .
interestingly , the major epitope of hnrnp a1 also comprises both rbds , which were also targeted by patients with rheumatoid arthritis and systemic lupus erythemathosus . taken together , these data confirm that rheumatoid arthritis , systemic lupus erythemathosus and mixed connective tissue disease are immunochemically linked by systemic autoimmunity to the functionally important rna - binding regions of hnrnp a / b proteins .
the observed trend brings the question of why so closely related and evolutionarily conserved rbd domains are differentially recognized by the autoantibodies .
we proposed recently ( 30 ) the overall folding patterns of these domains , and the protein antigenicity arising from these patterns is the determining factor .
the present study combined with previously determined sequences of u1a , hnrnp c , hnrnp a1 and drosophila sex lethal ( sxl ) protein structures reveals that they have the same fold , but have different placing of the second and fourth -strands .
individual rrms have preferences towards various rna sequences , due to differences in surface amino acids found outside the conserved rnp submotifs . in our opinion
in certain cases , the two rrms somehow act in concerted fashion to give rise to the overall rna- and antibody - binding characteristics , whereas in other cases the presence of only one rrm is sufficient for autoantibody and rna recognition .
thus , rbd1 itself bound strongly to rna fragments , however , the joining of rbd2 to rbd1 that increased the overall affinity indicats that rbd2 also strongly affects rna - binding .
to isolate the cdna , human liver and brain cdna libraries were screened by pcr using primers complementary to sequences in the 5- and 3-untranslated regions of the fetal brain ( fbrnp ) cdna , as described ( 15 ) .
screening of cdna expression library resulted in isolation of a clone , which is a member of a 2xrna - binding domain ( rbd)the glycine family of hnrnp proteins .
human hnrnp a3 polypeptide , highly homologous to the fetal brain cdna , as well as to hnrnps a1 and a2 . at the nucleotide level ,
the cdnas encoding rna - binding fragments of hnrnp a3 sequence were generated by pcr as deletion mutants , starting from the full - length human hnrnp a3 .
pcr mix was prepared by adding 660  l water , 220  l 10  mm dntp mix ( pharmacia biotech , uppsala , sweden ) , 220  l 10 cloned pfu polymerase buffer ( promega , madison , usa ) , and 122  l 25  mm mgcl2 for twice .
20  l of each oligonucleotide ( 100 pmol / ml ) was taken and added to 480  l water .
pcr reactions were run by taking 172  l of the pcr mix ( i. e. , 86  l per reaction ) , and mixing this amount with 12  l of both primers .
denaturation was achieved by incubating at 94 c for 30  sec , while annealing temperature was 52 c for 1  min .
the amplified fragments were cloned into ligation independent cloning ( lic ) vector ( novagen , madison , usa ) .
all the dna sequencings were performed by the vienna biocenter oligo team ( http://emb1.bcc.univie.ac.at/gem ) .
nucleotides and deduced protein sequences of previously determined rna - binding domains of rnps from various species and human hnrnp a3 were aligned employing clustalw programme ( http://www2.ebi.ac.uk/clustalw/ ; ref .
to isolate the cdna , human liver and brain cdna libraries were screened by pcr using primers complementary to sequences in the 5- and 3-untranslated regions of the fetal brain ( fbrnp ) cdna , as described ( 15 ) .
screening of cdna expression library resulted in isolation of a clone , which is a member of a 2xrna - binding domain ( rbd)the glycine family of hnrnp proteins .
human hnrnp a3 polypeptide , highly homologous to the fetal brain cdna , as well as to hnrnps a1 and a2 . at the nucleotide level ,
the cdnas encoding rna - binding fragments of hnrnp a3 sequence were generated by pcr as deletion mutants , starting from the full - length human hnrnp a3 .
pcr mix was prepared by adding 660  l water , 220  l 10  mm dntp mix ( pharmacia biotech , uppsala , sweden ) , 220  l 10 cloned pfu polymerase buffer ( promega , madison , usa ) , and 122  l 25  mm mgcl2 for twice .
20  l of each oligonucleotide ( 100 pmol / ml ) was taken and added to 480  l water .
pcr reactions were run by taking 172  l of the pcr mix ( i. e. , 86  l per reaction ) , and mixing this amount with 12  l of both primers .
denaturation was achieved by incubating at 94 c for 30  sec , while annealing temperature was 52 c for 1  min .
the amplified fragments were cloned into ligation independent cloning ( lic ) vector ( novagen , madison , usa ) .
all the dna sequencings were performed by the vienna biocenter oligo team ( http://emb1.bcc.univie.ac.at/gem ) .
nucleotides and deduced protein sequences of previously determined rna - binding domains of rnps from various species and human hnrnp a3 were aligned employing clustalw programme ( http://www2.ebi.ac.uk/clustalw/ ; ref .
 heterogeneous nuclear ribonucleoproteins ( hnrnps ) are spliceosomal macromolecular assemblages and thus actively participate in pre - mrna metabolism . 
 they are composed of evolutionarily conserved and tandemly repeated motifs , where both rna - binding and protein - protein recognition occur to achieve cellular activities . by yet unknown mechanisms , these ribonucleoprotein ( rnp ) particles are targeted by autoantibodies and hence play significant role in a variety of human systemic autoimmune diseases . 
 this feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity . 
 since rnp domain is one of the most conserved and widespread scaffolds , evolutionary analyses of these rna - binding domains can provide further clues on disease - specific epitope formation . 
 the study presented herein represents a sequence comparison of rna - recognition regions of recently cloned and characterized human hnrnp a3 with those of other relevant hnrnp a / b - type proteins . 
 their implications in human autoimmunity are particularly emphasized . 