hnRNP A1 Selectively Interacts Through its Gly-rich Domain with Different RNA-binding Proteins

The Microtubule-associated Protein Tumor Overexpressed Gene Binds to the RNA Trafficking Protein Heterogeneous Nuclear Ribonucleoprotein A2

“…HnRNP B1 and hnRNP A2 are splice isoforms of the same gene, and both proteins are expressed in oligodendrocytes (Kamma et al, 1999;Hatfield et al, 2002;Maggipinto et al, 2004). These results confirm those of Cartegni et al (1996) showing that hnRNP A2 binds to itself and to hnRNP B1 by yeast two-hybrid methodology.…”

“…HnRNP B1 and hnRNP A2 are splice isoforms of the same gene, and both proteins are expressed in oligodendrocytes (Kamma et al, 1999;Hatfield et al, 2002;Maggipinto et al, 2004). These results confirm those of Cartegni et al (1996) showing that hnRNP A2 binds to itself and to hnRNP B1 by yeast two-hybrid methodology.One of the unique clones, E3, contains the C-terminal third of a splice variant of ch-TOG, a protein previously identified as a MAP (X92474, gi 1045056, Charrasse et al, 1995) ( Figure 1B). The cDNA of a variant ch-TOG has previously been identified (D43948, gi 603950, Nagase et al, 1995).…”

Separable roles in vivo for the two RNA binding domains of a Drosophila A1-hnRNP homolog

“…In the Ddc mRNA splicing assay (Fig+ 2), no dominant negative effects on exon-skipping induction were seen when mutant HRB proteins were expressed+ Instead, two of the mutant proteins enhanced the exon-skipping activity+ These two proteins (R1-X-G and R1-R2-RS) are the only ones that bound reasonably well to multiple sites on polytene chromosomes as does the wildtype protein (Fig+ 5)+ The fact that enhancement, rather than suppression, was seen (even in the presence of an RS domain) is consistent with a nonspecific heteromultimerization that sterically blocks splice site selection+ Such a multimerization is also consistent with the self-association properties of the protein as revealed in the cytological assays, and with the ability of GRDs to associate with RS domains as well as with other GRD domains (Cartegni et al+, 1996)+ It is not clear whether the apparent involvement of RBD-1 in the enhancement phenomenon is because of a direct requirement for RBD-1 to bind this region of Ddc RNA or rather is an indirect effect of protein localization at chromosomal sites of transcription+ An alternative explanation for the results, that is, that excess RBD-1 is titrating a factor involved in splice-site selection for exon C of Ddc premRNA, seems unlikely because excess R1-X-G alone did not induce exon skipping+…”

“…In addition to direct binding of the GRD to RNA (Kumar et al+, 1990;Casas-Finet et al+, 1993), previous studies have also documented the self-association properties of the GRD of A1 hnRNP and of GRDs in general (Cartegni et al+, 1996), whether or not RNA is present (Casas-Finet et al+, 1993;Cartegni et al+, 1996)+ It is probably this self-association characteristic that underlies additional properties of the domain, such as its ability to confer cooperativity of binding Nadler et al+, 1991), to contribute to strand-annealing properties (Portman & Dreyfuss, 1994), and to contribute half of the free energy of binding of A1 hnRNP to RNA (Shamoo et al+, 1994)+ Various models have been proposed to explain the properties of these quasirepetitive domains (Steinert et al+, 1991;Pontius, 1993;Cartegni et al+, 1996) which have a semiregular spacing of aromatics (on average every 6 amino acids in A1 and every 9 amino acids in HRB87F/hrp36) and basic or polar amino acids embedded in glycines+ Although the models vary in particulars, they all predict an unusually flexible domain that can interact nonspecifically with RNA and with other GRDs via multiple, weak interactions that are easily disrupted and rearranged+ Pontius (1993) discusses how the unstructured repetitive nature of these domains is essential to their function in promoting association because multiple, transient interactions (both hydrophobic and electrostatic) can be formed regardless of the encounter orientation+ There is a positive correlation between GRD length and affinity to RNA (Buvoli et al+, 1990;Mayeda et al+, 1994) as well as affinity to other GRDs (Cartegni et al+, 1996)+ The critical importance of the aromatic residues in the interaction (Cartegni et al+, 1996) is in agreement with the suggestion that stacking of aromatics (with each other or with RNA bases) is involved+ Most of these previous studies have been done on the GRD of the A1 protein+ The GRD of HRB87F/hrp36 is ;40% longer than that of A1+ It has two RGG repeats near its N terminus and its middle region consists essentially of aromatics at intervals of 4-12 amino acids embedded in polar amides (20%) and glycine (60%)+…”

“…The large GRD at the C terminus contributes many of the unusual properties of A/B hnRNP binding+ It is ;120-200 residues long in proteins of this class and has a low sequence conservation, though a high conservation of glycine richness (40-50% gly), and a semiregular spacing of flexible, aromatic, and positively charged or polar amino acids (Cobianchi et al+, 1986) resulting in binding determinants along its entire length (Pontius, 1993)+ The GRD of HRB87F/hrp36 contains two RGG boxes (Kiledjian & Dreyfuss, 1992) near its N terminus and an M9-like nuclear shuttling signal near its C terminus+ In addition to binding RNA directly (Kumar et al+, 1990, Casas-Finet et al+, 1993, the GRD of A1 hnRNP is responsible for protein-protein interactions (independent of RNA) between proteins with a GRD (Casas-Finet et al+, 1993;Cartegni et al+, 1996) and for the cooperative binding of A1 to RNA Nadler et al+, 1991)+ We find that the GRD plays an important role in vivo, not only in nuclear localization of the protein, but also in specifying the quantitative deposition properties of the protein at sites of transcription and in protein sequestration at the 93D locus during heat shock+ Figure 1A shows the structure of the wild-type and mutant HRB87F/hrp36 proteins used in this study+ Transformed homozygous fly stocks were established in a wild-type background, each of which expressed a different mutant Hrb87F transgene under the control of the hsp70 promoter+ All of the proteins were epitope tagged at their N terminus with the Flag epitope so that they could be distinguished immunologically from the endogenous HRB87F protein after transformation into flies+ To abrogate the RNA binding properties of one or the other RBD, two highly conserved phenylalanines that occur in the RNP-1 box of the RBDs and participate directly in RNA binding (Merrill et al+, 1988;Oubridge et al+, 1994) were changed to aspartic acid residues, as was done previously for similar studies with human A1 hnRNP (Mayeda et al+, 1994)+ Although we have not done in vitro RNA-binding assays on these mutated proteins, there is ample evidence from previous studies that these changes to RNP-1 will severely compromise RNA-binding ability of the RBD as mediated via its RNA-binding b-sheet platform (e+g+, Mayeda et al+, 1994;Bouvet et al+, 1997;Deardorff & Sachs, 1997)+ This disruption of RNA-binding ability by mutation of conserved aromatics is also predicted by biophysical (Merrill et al+, 1988) and X-ray crystallographic analyses (Oubridge et al+, 1994)+ The phenylalanines were substituted by aspartic-acid residues, which are expected to remain solvent exposed without disrupting the structure of the domain, but unable to participate in the base-stacking interactions important to interaction of RNA with the RNA-binding platform of the RBD+ The wil...…”