Human Mitochondrial SUV3 and Polynucleotide Phosphorylase Form a 330-kDa Heteropentamer to Cooperatively Degrade Double-stranded RNA with a 3′-to-5′ Directionality

Abstract

Efficient turnover of unnecessary and misfolded RNAs is critical for maintaining the integrity and function of the mitochondria. The mitochondrial RNA degradosome of budding yeast (mtEXO) has been recently studied and characterized; yet no RNA degradation machinery has been identified in the mammalian mitochondria. In this communication, we demonstrated that purified human SUV3 (suppressor of Var1 3) dimer and polynucleotide phosphorylase (PNPase) trimer form a 330-kDa heteropentamer that is capable of efficiently degrading doublestranded RNA (dsRNA) substrates in the presence of ATP, a task the individual components cannot perform separately. The configuration of this complex is similar to that of the core complex of the E. coli RNA degradosome lacking RNase E but very different from that of the yeast mtEXO. The hSUV3-hPNPase complex prefers substrates containing a 3 overhang and degrades the RNA in a 3-to-5 directionality. Deleting a short stretch of amino acids (positions 510 -514) compromises the ability of hSUV3 to form a stable complex with hPNPase to degrade dsRNA substrates but does not affect its helicase activity. Furthermore, two additional hSUV3 mutants with abolished helicase activity because of disrupted ATPase or RNA binding activities were able to bind hPNPase. However, the resulting complexes failed to degrade dsRNA, suggesting that an intact helicase activity is essential for the complex to serve as an effective RNA degradosome. Taken together, these results strongly suggest that the complex of hSUV3-hPNPase is an integral entity for efficient degradation of structured RNA and may be the long sought RNA-degrading complex in the mammalian mitochondria.The current opinion on mitochondrial RNA degradation is largely based on our understanding of the Escherichia coli RNA degradosome and the yeast mitochondrial degradosome (mtEXO). The E. coli RNA degradosome consists of four components: RNase E, an endoribonuclease in which the C terminus serves as the scaffold of the multiprotein complex; PNPase, 4 an ambivalent enzyme that catalyzes 3Ј-to-5Ј phosphorolysis as well as 5Ј-to-3Ј polymerization of RNA; RhlB, a DEAD-box helicase; and enolase, a glycolytic enzyme (1-7). The 4-MDa multi-enzyme complex has been postulated to have a molar ratio of 4 RNase E:12 PNPase:4 RhlB:8 enolase (2, 4, 5, 8 -10). More recently, it has been reported that in the absence of RNase E, RhlB and PNPase can form a 380-kDa complex (2 RhlB:3 PNPase) to degrade dsRNA substrates (11-13). In budding yeast, an ATP-dependent DExH/D-box RNA helicase, Suv3p, and a 3Ј-to-5Ј directed exoribonuclease, Dss1, have been demonstrated to be the essential components of the mtEXO (14). In vitro, the two proteins have been shown to form a heterodimer that is capable of degrading dsRNA substrates containing a 3Ј overhang (15). Unlike its counterpart in the E. coli RNA degradosome, Dss1 does not form a trimeric ring structure but belongs to the RNR family of exoribonucleases that are mainly involved in rRNA maturation in the chloroplast of higher...

Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells

“…Apart from our study, a number of additional potential substrates for hPNPase old-35 have been identified, including small RNA and noncoding RNA. Very recently, Wang et al (17) described a model whereby mitochondrial hPNPase old-35 forms a heteropentameric complex with a helicase hSUV3 that functions in a coordinated manner to degrade dsRNA substrates in the presence of ATP. Mechanistically, helicase (hSUV3) unwinds the RNA substrate, and the exoribonuclease present in the same entity can efficiently degrade the structured RNA (18).…”

“…Apart from c-myc mRNA degradation, hPNPase old-35 is also involved in regulating the levels of several small noncoding RNAs (16). Very recently, Wang et al (17) reported that hPNPase old-35 , along with human mitochondrial SUV 3 (suppressor of Var1 3), degrades dsRNA in the 3′-to-5′ direction. Considering the above findings, it seemed plausible that hPNPase old-35 would have an impact on the other small RNAs, like miRNAs.…”

Uncoupling the Roles of the SUV3 Helicase in Maintenance of Mitochondrial Genome Stability and RNA Degradation

“…ATPase Assay, Gel Mobility Shift Assay, Helicase Assay, and Exoribonuclease Assay-Experimental procedures were adapted from a previous publication (40,41), and detailed procedures can be found in the supplemental Experimental Procedures. All DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (San Diego, CA) Sequences included T20DNA (5Ј-CAA ACT CTC TCT CTC TCA AC), 3WLRNA (5Ј-GUU GAG AGA GAG AGA GUU UGA GAG AGA GAG GUU UGA GAG AGA), and T22RNA (5Ј-CUC AAA CUC UCU CUC UCU CAA C).…”

“…Subsequent purification procedures using nickel beads were based on a previous publication (40). His-tagged DSS1 was co-transformed into Rosetta with pREP4 plasmid.…”

Conditional control of gene function by an invertible gene trap in zebrafish

“…It is an essential component of the mitochondrial degradosome complex that regulates mtRNA decay and mRNA surveillance (19). Human SUPV3L1 is important for maintaining mitochondrial homeostasis (20) and has been shown to degrade double-stranded RNA (21). However, SUPV3L1 also appears to function in the cytoplasm, interacting with the WRN and BLM DNA helicases to suppress mitotic homologous recombination (13).…”