CRYSTAL-STRUCTURE OF HUMAN UP1, THE DOMAIN OF HNRNP-A1 THAT CONTAINS 2 RNA-RECOGNITION MOTIFS

Background: Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is one of the most abundant core proteins of hnRNP complexes in metazoan nucl ei. It behaves as a global regulator of alternative pre-mRNA splicing by antagonizing the activities of several serine/arginine-rich splicin g factors (SR proteins), resulting in the activation of distal alterna tive 5' splice sites and skipping of optional exons. Purified hnRNP A1 has nucleic acid annealing activity. The protein also shuttles contin uously between the nucleus and the cytoplasm, a process mediated by si gnals within its C-terminal glycine-rich domain. The N-terminal region of human hnRNP A1, termed unwinding protein 1 (UP1), contains two RNA -recognition motifs (RRMs), RRM1 and RRM2. Understanding the structura l elements by which hnRNP A1 interacts with RNA will have broad implic ations for studies of RNA processing. Results: The crystal structure o f UP1 has been determined to 1.9 Angstrom resolution. Each RRM indepen dently adopts the characteristic RRM fold, consisting of a four-strand ed antiparallel beta-pleated sheet and two of helices packed on one si de of the beta sheet. The two RRMs are antiparallel and held in close contact, mainly by two Arg-Asp ion pairs. As a result, the two tour-st randed beta sheets are brought together to form an extended RNA-bindin g surface. A segment of the linker connecting the two RRMs is flexible in the absence of bound RNA, but the general location of the linker s uggests that it can make direct contacts with RNA. Comparison with oth er RRM structures indicates that a short 3(10) helix, found immediatel y N-terminal to the first beta strand in RRM1, may interact with RNA d irectly. Conclusions: The RRM is one of the most common and best chara cterized RNA-binding motifs, in certain cases, one RRM is sufficient f or sequence-specific and high affinity RNA binding; but in other cases , synergy between several RRMs within a single protein is required. Th is study shows how two RRMs are organized in a single polypeptide. The two independently folded RRMs in UP1 are held together in a fixed geo metry, enabling the two RRMs to function as a single entity in binding RNA, and so explaining the synergy between the RRMs, The UP1 structur e also suggests that residues which lie outside of the RRMs can make p otentially important interactions with RNA.