Characterization of 3′hExo, a 3′ Exonuclease Specifically Interacting with the 3′ End of Histone mRNA

Abstract

The 3 end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3 exonuclease designated 3hExo. The sequence-specific interaction suggests that 3hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3hExo is a member of the DEDDh family of 3 exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3hExo binds with the highest affinity to the wild-type 3 end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3hExo has only residual, if any, 3 exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3hExo requires 2 and 3 hydroxyl groups at the last nucleotide. 3hExo removes 3 overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.Mammalian histone mRNAs end with a highly conserved and unique stem-loop structure followed by a single-stranded ACCCA sequence (1). The 3Ј end of histone mRNAs is specifically recognized by two proteins, the stem-loop-binding protein (SLBP) and 3ЈhExo (2). Binding of SLBP requires the nucleotides at the 5Ј side of the stem-loop, whereas binding of 3ЈhExo requires the single-stranded ACCCA at the 3Ј end. In addition, binding of each protein requires specific nucleotides in the stem and the loop. SLBP and 3ЈhExo can bind the 3Ј end of histone mRNA either individually or simultaneously, forming a ternary complex (2). SLBP stimulates binding of 3ЈhExo to the stem-loop and allows 3ЈhExo to form a complex with suboptimal RNA targets, raising the possibility that the two proteins make direct contact with each other in the ternary complex (2). Binding of SLBP to the stem-loop in histone mRNA precursors is required for formation of the correct 3Ј end of histone mRNAs in the nucleus (3). SLBP bound to the stem-loop accompanies mature histone mRNA to the cytoplasm, where it stimulates histone mRNA translation (4).The half-life of histone mRNAs is greatly reduced in response to completion or inhibition of DNA replication, resulting in rapid disappearance of histone mRNAs from the cytoplasm and cessation of histone production (5). The stemloop structure is both necessary and sufficient for the selective degradation of histone mRNA and confers the same type of regulation on other mRNAs when introduced at their 3Ј end (6). Sequence-specific and ...

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

“…Taken together, our findings strongly support the conclusion that Eri1 catalyzes the removal of unpaired 3′ nucleotides from the end of an already-formed 5.8S-28S rRNA duplex. This characteristic activity of the enzyme is consistent with previous observations that Eri1 activity is enhanced on free 3′ overhangs of siRNA duplexes compared with single-stranded RNA or RNA duplexes 1,11 .…”

“…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.…”

“…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

“…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.…”

“…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).…”