Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Abstract

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-␤-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in F o F 1 -ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

Polyadenylation in Arabidopsis and Chlamydomonas organelles: the input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase

“…In addition to these two enzymes, in archaeal species in which polyadenylation takes place, the archaeal exosome complex, which is very similar to PNPase, is responsible for polyadenylation, producing heteropolymeric tails (Portnoy et al, 2005). Indeed, PNPase is present in every bacterium and organelle where polyadenylation takes place, excluding the mitochondria of trypanosomes, although in mammalian mitochondria it is located in the intermembrane space, physically separated from mtRNA (Chen et al, 2006(Chen et al, , 2007. In several bacteria such as cyanobacteria and the Grampositive S. coelicolor, there is no Ntr-PAP, and PNPase is solely responsible for polyadenylation activity, producing heteropolymeric tails (Rott et al, 2003;Sohlberg et al, 2003).…”

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

“…Human PNPase was identified in an overlapping pathway screen to discover genes displaying coordinated expression as a consequence of terminal differentiation and senescence of melanoma cells [15][16][17]. In human cells PNPase is mostly located in the mitochondrial intermembrane space and may not play the major role in the processing and degradation of RNA that it plays in bacteria, chloroplasts and plant mitochondria [18,19].…”

Crystal structure of Escherichia coli PNPase: Central channel residues are involved in processive RNA degradation

“…Mammalian PNPase is intriguingly located in the mitochondrial intermembrane space where no RNA is present (Piwowarski et al 2003). Recent studies suggest that human PNPase has a crucial role in maintaining mitochondrial homeostasis and is induced by type I interferons, likely playing a role in antiviral defense and cellular senescence (Leszczyniecka et al 2003;Chen et al 2006Chen et al , 2007French et al 2007). However, the molecular basis for the mammalian PNPase regulation of these fundamental physiological events is largely unknown.…”