A 3′ Exonuclease that Specifically Interacts with the 3′ End of Histone mRNA

Abstract

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.

Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA processing

“…Previous reports demonstrated that the RNA binding function of the SAP and linker domains of Eri1 are essential for sequence-specific binding to the histone mRNA stem loop and its conserved 3′-terminal ACCCA sequence 6,11 . In our experiments, the SAP domain and linker sequences were important for stable interaction with the 5.8S rRNA at low or endogenous expression levels.…”

“…Also known as Thex1 in some species, Eri1 and its ability to inhibit short interfering RNA (siRNA)-directed gene silencing is evolutionarily conserved from Schizosaccharomyces pombe to humans [1][2][3][4] . Because Eri1 encodes a DEDDh-type exonuclease with RNase activity toward the 3′ ends of RNA in vitro 1,[4][5][6] , it was proposed that Eri1 counteracts the RNAi pathway by degrading siRNAs 7 . Alternatively, Eri1 may inhibit RNAi indirectly by sequestering factors involved in siRNA processing into a different protein complex [8][9][10] .…”

“…Basic amino acid residues in the Eri1 SAP (SAF-A/B, Acinus and PIAS) domain and interdomain linker are necessary for in vitro binding to RNA oligonucleotide mimics of a stem-loop structure at the 3′ end of histone mRNAs 6,11 . Thus, Eri1 has been implicated in the regulation of both siRNAs and histone mRNAs.…”

The exonuclease ERI-1 has a conserved dual role in 5.8S rRNA processing and RNAi

“…In the fission yeast S. pombe loss of Eri1 causes increased levels of small interfering RNAs (siRNAs) corresponding to centromeric repeats and a concomitant increase in RNAi-dependent heterochromatin formation at these genomic loci 2 . Analysis of ERI-1 in C. elegans, human and fission yeast has shown that it can degrade the 3′ end of siRNAs and histone mRNAs in vitro [1][2][3][4] , but in vivo substrates for this conserved enzyme are poorly understood.In the course of the analysis of RNAs isolated from the eri-1 null mutant, we observed that the 5.8S rRNA in an eri-1 worm is longer than wild-type 5.8S rRNA (Fig. 1a).…”

“…The mature 5.8S, 18S, and 25-28S rRNAs in eukaryotes are generated from a 35S-47S precursor RNA via a series of processing steps mediated by multiple nucleases 5 . The activity of ERI-1 as a 3′ to 5′ exonuclease [1][2][3][4] suggests that the longer 5.8S rRNA is due to an extension of the 3′ end. RNase H cleavage and 3′ end cloning on the 5.8S rRNA of wild-type and mutant C. elegans indicated that all eri-1 5.8S rRNA is at least 1 nucleotide longer than the wild-type 5.8S rRNA specifically at the 3′ end, with a substantial fraction of eri-1 5.8S rRNA containing 2 to 4 additional nucleotides ( Supplementary Fig.…”