Crystal structure of human polynucleotide phosphorylase: insights into its domain function in RNA binding and degradation

Lin, Chia‐Liang; Wang, Yi Ting; Yang, Wen-Chieh; Hsiao, Y.-Y.; Yuan, Hanna S.

doi:10.1093/nar/gkr1281

Cited by 63 publications

(92 citation statements)

References 51 publications

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“…(III) En route, the RNA strand makes nonequivalent contacts with the KH domains (16), in addition to making contact within a putative core binding site (red asterisks). long 3= overhang to thread an RNA into the cleavage channel of human PNPase is also consistent with the simultaneous engagement of RNA 3= termini by both S1 and KH domains and the "hands on a rope" model (45). We predict that KH domains with enhanced affinity for RNA may prove inhibitory to PNPase by trapping RNA on the surface and inhibiting ratcheting.…”

Section: Figsupporting

confidence: 74%

“…From recent structural determinations of PNPase and its homologues, it appears that the KH and S1 domains communicate with the conformationally dynamic catalytic core (15,19,45). Accordingly, an additional role for the KH domain may be the modulation of the size of the narrow "gateway" aperture to regulate substrate accommodation within the channel upon RNA binding.…”

Section: Figmentioning

confidence: 99%

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S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

et al. 2013

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To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between ␤-galactosidase levels reflecting autoregulation and apparent K D values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase. Polynucleotide phosphorylase (PNPase) is a conserved, widely distributed phosphorolytic 3=-5= exoribonuclease that may also function under some circumstances as a template-independent RNA polymerase (1; reviewed in reference 2). Although it is not essential, deletions of its gene (pnp) are synthetically lethal when either RNase II or RNase R, both of which are hydrolytic 3=-5= exoribonucleases, is deficient (3-5). Strains deficient in PNPase are also sensitive to cold shock and other stresses (6-9). Thus, PNPase is believed to play significant roles in mRNA turnover and other aspects of RNA processing and metabolism (4, 10-12). Partial or full structures of PNPase from several microorganisms (13-16), as well as structures of the related archaeal exosome (17, 18), have shed considerable light on its mechanism of action. Bacterial PNPase is composed of three identical subunits. Each subunit consists of two tandem globular domains (residues 8 to 210 and 312 to 541; see Fig. 1a) derived from RNase PH that form a core whose central channel is accessible from both the upper and lower surfaces of the core (14,19). Each subunit also contains a C-terminal extension that consists of two additional small domains, KH and S1 (residues 551 to 591 and 622 to 691, respectively; Fig. 1), which are positioned on the upper surface of the core. The KH and S1 domains have been implicated in autoregulation (20), in resistance to cold shock (7), in pathogenesis (21), and in substrate binding (22). Although a solution structure of the S1 domain from Escherichia coli PNPase has been available (13), the positions of the S1 and KH domains relative to the core have been elucidated only recently from the structure of PNPase from Caulobacter crescentus (16). The S1 and KH domains do not appear to be required for the enzymatic activity of PNPase (20,22,23). Nonetheless, deletion of the S1, the KH or both domains results in significant loss of RNA binding and inefficient enzymatic turnover (22).PNPase from E. coli and other organisms exhibits strong autoregulation...

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Section: Figsupporting

confidence: 74%

Section: Figmentioning

confidence: 99%

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

et al. 2013

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“…Utilizing PNPase for RNA import into the mitochondrion requires the presence of a specific stem-loop secondary structure in the target RNA [57,92]. Interestingly, Barrey et al found that pre-miRNA-302a and pre-let-7b are predicted to fold into these stem-loop configurations, leading to the assumption that PNPase could be critical for the import of these pre-miRNAs into the mitochondrion [5].…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, a major determining factor between RNA degradation or transport by PNPase seems to be the length of the RNA species’ 3′ overhang. Wang et al showed that an 8 nucleotide (nt) 3′ overhang of a mRNA species resulted in degradation while a 0–2 nt 3′ overhang led to transport [57,84]. Other studies suggest that the machinery facilitating protein import into the mitochondria may also contribute to RNA import [3,36,54,75,78,81,82].…”

Section: Introductionmentioning

confidence: 99%

Exploring the mitochondrial microRNA import pathway through Polynucleotide Phosphorylase (PNPase)

Shepherd

Hathaway

Pinti

et al. 2017

Journal of Molecular and Cellular Cardiology

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Cardiovascular disease is the primary cause of mortality for individuals with type 2 diabetes mellitus. During the diabetic condition, cardiovascular dysfunction can be partially attributed to molecular changes in the tissue, including alterations in microRNA (miRNA) interactions. MiRNAs have been reported in the mitochondrion and their presence may influence cellular bioenergetics, creating decrements in functional capacity. In this study, we examined the roles of Argonaute 2 (Ago2), a protein associated with cytosolic and mitochondrial miRNAs, and Polynucleotide Phosphorylase (PNPase), a protein found in the inner membrane space of the mitochondrion, to determine their role in mitochondrial miRNA import. In cardiac tissue from human and mouse models of type 2 diabetes mellitus, Ago2 protein levels were unchanged while PNPase protein expression levels were increased; also, there was an increase in the association between both proteins in the diabetic state. MiRNA-378 was found to be significantly increased in db/db mice, leading to decrements in ATP6 levels and ATP synthase activity, which was also exhibited when overexpressing PNPase in HL-1 cardiomyocytes and in HL-1 cells with stable miRNA-378 overexpression (HL-1-378). To assess potential therapeutic interventions, flow cytometry evaluated the capacity for targeting miRNA-378 species in mitochondria through antimiR treatment, revealing miRNA-378 level-dependent inhibition. Our study establishes PNPase as a contributor to mitochondrial miRNA import through the transport of miRNA-378, which may regulate bioenergetics during type 2 diabetes mellitus. Further, our data provide evidence that manipulation of PNPase levels may enhance the delivery of antimiR therapeutics to mitochondria in physiological and pathological conditions.

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“…Single-stranded miRNA can be degraded by the 5 0 -3 0 exoribonuclease XRN2 or 3 0 -5 0 exoribonuclease human polynucleotide phosphorylase [50,51]. Previous studies have shown that the binding of pre-miRNAs to protein components such as MCPIP1 can facilitate the process of pre-miRNA degradation [52].…”

Section: Dna Damage Modulates Mirna Degradationmentioning

confidence: 99%

Non-coding RNAs: An emerging player in DNA damage response

Zhang

Peng

2015

Mutation Research/Reviews in Mutation Research

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Crystal structure of human polynucleotide phosphorylase: insights into its domain function in RNA binding and degradation

Cited by 63 publications

References 51 publications

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

Exploring the mitochondrial microRNA import pathway through Polynucleotide Phosphorylase (PNPase)

Non-coding RNAs: An emerging player in DNA damage response

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