Probing the substrate specificity of the bacterial
Pnkp/Hen1 RNA repair system using synthetic RNAs
CAN ZHANG,1CHIO MUI CHAN,1,2PEI WANG, and RAVEN H. HUANG3
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to
counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago.
Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-
cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial
Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/
Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms
of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1
RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred
RNA substrates of the Pnkp/Hen1 repair system is proposed.
Keywords: RNA repair; Pnkp/Hen1; substrate specificity; RNA damage; ribotoxin
RNA in living cells is highly susceptible to damage, pri-
marily due to breakage of the phosphodiester backbone.
Phosphodiester bond cleavages are usually triggered by
ribonucleases, assisted by the adjacent 29 OH group in RNA
at the site of breakage, generating 29,39-cyclic phosphate at
the 39 end and 59 OH at the 59 end in most cases. Most
RNA breakdown is part of normal metabolism, carried out
by endogenous ribonucleases. However, cleavage of essen-
tial RNAs is exploited by some organisms as a strategy for
cell killing. The agents responsible for the RNA cleavage are
proteins called ribotoxins, which are usually site-specific
ribonucleases. The ribotoxins identified to date kill cells by
targeting rRNAs and tRNAs involved in protein translation.
Known ribotoxins targeting rRNAs are sarcin (Endo and
Wool 1982), restrictocin (Fando et al. 1985), colicin E3
(Bowman et al. 1971), and Biota orientalis ribonuclease (Xu
et al. 2004). Several ribotoxins such as colicin E5 (Ogawa
et al. 1999), colicin D (Tomita et al. 2000), and Kluyver-
omyces lactis g-toxin (Lu et al. 2005) are tRNA-specific
ribotoxins, which make a single cut in the anticodon loop
of a specific tRNA. In addition to the external ribotoxins
that invade cells and damage RNA, a cell sometimes pro-
duces its own site-specific ribonucleases to regulate gene
expression and cell fate. For example, when infected by
bacteriophage T4, some strains of Escherichia coli activate
ribonuclease PrrC, which cleaves tRNALysat the wobble
position in the anticodon loop (Amitsur et al. 1987). RelE
is activated to cleave mRNAs at the ribosome A site to re-
strict global protein production when a cell is starved
(Pedersen et al. 2003; Neubauer et al. 2009). More recently,
VapC was shown to have the same effect as RelE but by
cleaving the initiator tRNAfMetin the anticodon loop
(Winther and Gerdes 2011). In eukaryotic organisms, stress
causes significant RNA damage, which may involve ribo-
nucleases (Lee and Collins 2005; Thompson et al. 2008;
Yamasaki et al. 2009).
Two proteins from bacteriophage T4, Pnkp (polynucle-
otide kinase-phosphatase) and Rnl1 (RNA ligase 1), coun-
teract the suicidal action of PrrC in infected E. coli by
RNA repair (Amitsur et al. 1987). Specifically, bifunctional
T4Pnkp hydrolyzes the 29,39-cyclic phosphate at the 39 end
and adds a phosphate group at the 59 end of the cleaved
tRNALys. Subsequently, T4Rnl1 ligates the two processed
1These authors contributed equally to this work.
2Present address: Division of Environmental and Biomolecular Sys-
tems, Oregon Health and Science University, Beaverton, OR 97006, USA.
Article published online ahead of print. Article and publication date are
RNA (2012), 18:335–344. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2012 RNA Society.
ends to restore the tRNALysto its original form. Thus, the
RNA repair carried out by bacteriophage T4 precludes
disruption of T4 late translation and thus rescues the
Recently, we found that two bacterial proteins repair
ribotoxin-cleaved tRNAs in vitro (Chan et al. 2009b). The
two proteins, bacterial Pnkp and Hen1, physically interact
with each other, forming a heterotetramer in vitro. The
bacterial Pnkp from Clostridium thermocellum was first
characterized by Martins and Shuman (2005), who showed
59 kinase, 29,39 phosphatase, and adenylyltransferase activ-
ities. Interestingly, Pnkp alone could not repair a cleaved
RNA (Keppetipola et al. 2007). We demonstrated that RNA
repair requires both Pnkp and Hen1 (Chan et al. 2009b).
The Pnkp/Hen1 RNA repair system is distinct from the
T4Pnkp/Rnl1 system due to the involvement of a fourth
enzymatic activity, 29-O-methyltransferase, which is located
at the C terminus of bacterial Hen1. During RNA repair,
bacterial Hen1 carries out 29-O-methylation at the 39
cleavage end after dephosphorylation of the 29,39-cyclic
phosphate but before ligation of the two cleavage ends
(Chan et al. 2009b). Due to the 29-O-methylation at the
junction of repair, the same RNA damage can no longer
occur in the repaired RNA.
In contrast to the extensively characterized T4 system, we
know much less about the more recently discovered bacte-
rial Pnkp/Hen1 RNA repair system. Prior studies from the
Shuman laboratory and our laboratory have focused on in
vitro characterization, either of the Pnkp/Hen1 complex
(Chan et al. 2009b) or of components of the complex
(Martins and Shuman 2005; Keppetipola et al. 2007; Chan
et al. 2009a; Jain and Shuman 2010, 2011). Perhaps the
biggest gap is the lack of knowledge about the in vivo RNA
substrate(s) of Pnkp/Hen1 and the biological effect of RNA
repair in strains containing the Pnkp/Hen1 RNA repair
system. If RNA damage is caused internally in bacterial
species possessing the Pnkp/Hen1 RNA repair system, it is
possible that an in vivo study on one of these bacterial
species would provide insight into RNA damage and repair.
On the other hand, it is possible that RNA damage in
these bacteria could be caused by external agents such as
ribotoxins released from other surrounding organisms. If
that were the case, then RNA repair would only occur when
these bacteria live under conditions that induce such
damage, which might not be readily reproduced in a labo-
Here we present a comprehensive in vitro RNA repair
study using Pnkp/Hen1 from Anabaena variabilis (AvaPnkp/
Hen1) and a variety of RNA substrates, with the aim of
providing insight into the likely in vivo RNA substrates of
the Pnkp/Hen1 RNA repair system. We found that PnkP/
Hen1 may exhibit broader substrate specificity than T4PnkP/
Rnl1. Based on the studies presented here, the likely in vivo
RNA substrates of Pnkp/Hen1 and the possible biological
roles of Pnkp/Hen1 are discussed.
AvaPnkp/Hen1 efficiently repairs tRNA
Prior to finding the RNA repair capabilities of bacterial
Pnkp/Hen1, several RNA ligase enzymes, with implications
for RNA editing or repair, have been biochemically char-
acterized (Amitsur et al. 1987; Ho and Shuman 2002;
Simpson et al. 2003; Martins and Shuman 2004a,b). Among
them, the T4Pnkp/Rnl1 is the most extensively character-
ized as an RNA repair system. The in vivo RNA repair
target of the T4 system was well defined: to repair E. coli
tRNALyscleaved by the endogenous ribonuclease PrrC.
Furthermore, the T4Pnkp/Rnl1 repair system appears to
have evolved to optimize RNA repair of the cleaved tRNA;
this has been suggested by respective T4Pnkp/tRNA and
T4Rnl1/tRNA docking experiments (Galburt et al. 2002; El
Omari et al. 2006), as well as a subsequent study by Shuman
and coworkers (Nandakumar et al. 2008). Using both the
cleaved full-length and tRNA deletion mutants, Shuman and
coworkers were able to demonstrate that the efficiency of
repair for the cleaved full-length tRNA is significantly higher
than the ones for the cleaved tRNA deletion mutants
(Nandakumar et al. 2008).
Considering the possibility that like the T4 RNA repair
system, the cleaved tRNAs could also be the natural repair
targets of the bacterial Pnkp/Hen1 RNA repair system, we
performed similar experiments with the AvaPnkp/Hen1
proteins. The cleaved tRNAAspand its deletion derivatives
were our first choice for the experiments because, like the
E. coli tRNALyscleaved by PrrC, these tRNAs are cleaved by
colicin E5 at a position near the wobble nucleotide (Fig. 1).
The cleaved full-length tRNAAspwas efficiently repaired by
AvaPnkp/Hen1, with a repair yield as high as 65% after 60
min of repair reaction (Fig. 2A,C, labeled with tRNAAsp).
Deletion of the D stem–loop in tRNAAspresulted in a
significantly worse RNA substrate, with a repair yield of
15% after 60 min of repair reaction (Fig. 2A,C, labeled with
tRNAAsp-DD). Deletion of the TCC stem–loop was not as
bad, with a final repair yield of 40% (Fig. 2A,C, labeled with
tRNAAsp-DT). Also, AvaPnkp/Hen1 was still able to repair
a tRNAAspdeletion mutant with both D and TCC stem–
loops deleted, with a repair yield comparable to the de-
letion of D stem–loop alone (Fig. 2A,C, labeled with
tRNAAsp-DDT). In addition to the repaired tRNAAsp-
DDT, some ligation products of higher molecular weight
(Fig. 2A, marked with *) were also produced with the cleaved
tRNAAsp-DDT as the substrate. We noticed that tRNAAsp-
DDT-39 has five continuous Gs (Table 1). Therefore, it is
possible that repair of abnormal annealed RNA substrates
resulted in production of additional ligation products.
Next, we carried out RNA repair of the cleaved tRNAArg
and its deletion derivatives (Fig. 2B,D). tRNAArgand its
deletion derivatives were cleaved by colicin D at a position
Zhang et al.
RNA, Vol. 18, No. 2
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