Mapping domains of ARS2 critical for its RNA decay capacity

Abstract

<jats:title>Abstract</jats:title> <jats:p>ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5′cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.</jats:p>

Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex

“…This is consistent with the demonstration that the hARS2 construct corresponding to the C-terminal leg (628-876) is a major determinant in triggering RNA decay of endogenous PROMPTs transcripts and in tethering assays 40 . Indeed, in tethering assays, the ARS2 activity was reduced upon siRNA silencing of hZFC3H1, but not of ZC3H18 nor ZCCHC8 40 . Moreover, either a K719A, K722A, K734A triple mutation, or the single K719A mutation, in the C-terminal leg of ARS2 inhibited its RNA decay triggering activity 40 .…”

“…Correspondingly, we show that hARS2 and hZFC3H1 interact and the E23R mutation in hZFC3H1 (equivalent to E32R in Red1) is sufficient to disrupt the complex. This is consistent with the demonstration that the hARS2 construct corresponding to the C-terminal leg (628-876) is a major determinant in triggering RNA decay of endogenous PROMPTs transcripts and in tethering assays 40 . Indeed, in tethering assays, the ARS2 activity was reduced upon siRNA silencing of hZFC3H1, but not of ZC3H18 nor ZCCHC8 40 .…”

“…Moreover, either a K719A, K722A, K734A triple mutation, or the single K719A mutation, in the Cterminal leg of ARS2 inhibited its RNA decay triggering activity 40 . Here, we show that residues Lys719 and Lys722 are directly involved in recognizing the Red1/ZFC3H1 EDGEI motif.…”

Cytoplasmic Switch of ARS2 Isoforms Promotes Nonsense-Mediated mRNA Decay and Arsenic Sensitivity

“…To understand how ARS2 isoforms differentially regulate NMD, we first asked whether the inhibitor role of ARS2n on the NMD pathway was dependent on its nuclear localization. We narrowed the nuclear localization signal region from 105 amino acids 48 to 12, and generated an ARS2n mutant which lacks amino acids (73-LSPPQKRMRRDW-84). As shown in Fig.…”

Stealing the Show: KSHV Hijacks Host RNA Regulatory Pathways to Promote Infection

“…In the first set of experiments, they discovered that depletion of the protein ARS2-a key factor in both decay pathways-led to stabilization of both PAN∆ENE and ORF59 in the absence of ORF57. ARS2 contributes to these decay pathways by recruiting an RNA helicase protein MTR4 (hMTR4) and an exosome cofactor [60]. Knockdown of hMTR4 was able to restore transcript levels of both PAN∆ENE and ORF59 in absence of ORF57, indicating that ORF57 protects transcripts from hMTR4-mediated decay.…”