FC0103
ALYREF  -  ORF57, RNA

Biological function
ALYREF functions as an export adaptor as part of cellular transcription-export TREX, via binding mRNA, and also interacting with NXF1, which is essential for the export of cellular mRNA via the NXF1 pathway. ALYREF associates with the 5’ end of cellular mRNAs during splicing. ALYREF may be recruited by viral adaptors to stabilize the viral nuclear RNAs independently of their export.

Domain organization/sequence features
ALYREF has three domains: a central folded RRM domain flanked by two largely flexible multifunctional N- and C-terminal domains. ALYREF primarily uses its N-terminal flexible arginine-rich region for interaction with NXF1; this region closely overlaps with the RNA binding site. The arginines within this region become methylated, which reduces its RNA binding activity and may serve as a control mechanism for RNA displacement from ALYREF to NXF1.

Structural evidence
NMR signal intensity of the N-terminal region aa22–48 of ALYREF does not reduce significantly upon RNA binding in the presence of ORF57, suggesting that this interaction is relatively transient. As evidenced by NMR signal shapes and lack of RNA-protein NOEs the N-terminal regions of both ORF57 and ALYREF in contact with RNA retain significant flexibility when the ternary RNA-ORF57-ALYREF complex is formed. The main NXF1-binding region of ALYREF aa15–36 partially overlaps with region involved in viral mRNA binding, and remains sufficiently exposed.

Biochemical evidence
A very weak non-specific binding of RNA to ALYREF was observed with estimated K_D of 50 mM. K_D of RNA binding to the ORF57-ALYREF is 1.55±0.24 μM, indicating a cooperative behavior of ALYREF and ORF57. K_D of ALYREF for ORF57-RNA is 0.52 μM, 2 orders of magnitude higher than that of the binary complex with RNA. K_D of ORF57^8-120 and ALYREF^1-155 is 2.56±0.20 μM, also consistent with the cooperativity of the ternary complex.

Structure/Mechanism
ALYREF partially displaces the viral RNA initially bound specifically to ORF57, but retains it within the complex. In the ternary complex ORF57 aa106–120 directly interacts with the ALYREF RRM, whereas flexible flanking regions of ALYREF (aa24–48) and ORF57 (aa81–92), and to lesser extent, parts of helix 2 of the ALYREF RRM, jointly keep hold of the viral RNA molecule. Thus RNA is partially displaced from its binding site on ORF57 by ALYREF, but is retained in the complex by the synergistic action of flanking flexible regions of both ALYREF and ORF57.

Mechanism category
tethering

Significance
Fuzziness of the ternary RNA-ORF57-ALYREF complex enables combination of competitive and cooperative binding of partially-overlapping multifunctional binding sites for the assembly and disassembly of mRNA nuclear export complexes thus facilitating a key molecular transfer event in herpesvirus highjacking of the host nuclear export pathway.