Characterization of the mammalian RNA exonuclease 5/NEF-sp as a testis-specific nuclear 3′ → 5′ exoribonuclease

Abstract

Ribonucleases catalyze maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp (RNA exonuclease 5 [REXO5] or LOC81691) as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3 ′ ′ ′ ′ ′ → 5 ′ ′ ′ ′ ′ exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length. The presence of a 2 ′ ′ ′ ′ ′ -O-methyl modification at the 3 ′ ′ ′ ′ ′ end of the RNA substrate is inhibitory. Ectopically expressed NEF-sp localizes to the nucleolar/nuclear compartment in mammalian cell cultures and this is dependent on an amino-terminal nuclear localization signal. Finally, mice lacking NEF-sp are viable and display normal fertility, likely indicating overlapping functions with other nucleases. Taken together, our study provides the first biochemical and genetic exploration of the role of the NEF-sp exoribonuclease in the mammalian genome.

The Conserved RNA Exonuclease Rexo5 Is Required for 3′ End Maturation of 28S rRNA, 5S rRNA, and snoRNAs

“…Further supporting a conserved role of Rexo5 in metazoan rRNA biogenesis, human REXO5 also exhibits nucleolar localization and RNA exonuclease activity (Figure S2J) (Silva et al, 2017). Future investigation of the vertebrate Rexo5 homologs, as well as other Rexo family members, will clarify their role in nuclear maturation of rRNAs.…”

“…The enzymatic activity of Rexo5 wt was inhibited by EDTA, establishing a Mg 2+ -dependent 3′-5′ RNA exonuclease activity. Human REXO5 was recently biochemically isolated and shown to have 3′-5′ specific ssRNA exonuclease activity in vitro, which was inhibited by 2′-O-methylation of the substrate 3′ end (Silva et al, 2017). …”

Pre-Ribosomal RNA Processing in Human Cells: From Mechanisms to Congenital Diseases

“…This exonuclease is evolutionary conserved, and its human ortholog is located in the nucleolus [35,93], which supports a similar role in human 5S* RNA processing. However, in mouse, Rexo5 is not essential to viability and fertility, and it does not concentrate in the nucleolus [93]. After processing of the 3′ end, the 5S rRNA is associated with the ribosomal protein L5 (RPL5) in a complex that does not include La [94].…”

“…The 5S* RNA 3′ end is processed by a 3′–5′ exonuclease, which was recently identified as REXO5 in Drosophila [35]. This exonuclease is evolutionary conserved, and its human ortholog is located in the nucleolus [35,93], which supports a similar role in human 5S* RNA processing. However, in mouse, Rexo5 is not essential to viability and fertility, and it does not concentrate in the nucleolus [93].…”

A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

“…(3,4)). There are also several yet uncharacterised proteins harbouring predicted nuclease domains in mammalian genomes (5)(6)(7)(8).…”