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A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi
Takashi Kameda, Kenji Ikegami, Yang Liu, Kunihiko Terada, and Toshihiro Sugiyamundefined
Department of Biochemistry, Akita University School of Medicine 1-1-1 Hondo, Akita 010-8543, Japan
Received 12 January 2004
Abstract
RNA interference (RNAi) has been attracting a great deal of attention. This pathway is highly conserved among most eukaryotes
and believed to be important for antiviral reactions and epigenetic gene regulation. Because a temperature-sensitive RNAi was
reported in both plant and insect systems, suggesting its evolutional conservation, we analyzed the effect of different temperatures on
mammalian RNAi, targeting the ectopic gene expression, and detected suppression at hypothermic temperatures. This phenomenon
could be critical and useful to control ectopic and internal gene expressions by RNAi.
2004 Elsevier Inc. All rights reserved.
Keywords: Mammalian RNAi; Temperature biology
RNAi is a nucleotide sequence-specific RNA degradation
pathway, reactive to double-stranded RNA [1–
3]. A pathway homologous to RNAi is highly conserved
among most eukaryotes. While the biochemical mechanism
of RNAi is yet to be fully revealed, this pathway
is composed of several steps, including the production
of 21–25 nt short-interfering RNA (siRNA) from
dsRNA by the RNase III-like enzyme complex (DICER),
and the sequence-specific degradation of the
target RNA by the nuclease complex (RISC). In some
organisms, amplification of the siRNA by the RNAdependent
RNA polymerase (RdRP) and intercellular
transport of the siRNA (systemic pathway) intensify the
reaction. The RNAi in eukaryotes is thought to be an
anti-viral reaction as well as an epigenetic chromosomal
regulator, involved in the suppression of internal
transposon [1–5].
Most biochemical reactions are sensitive to temperature.
In Drosophila, temperature-dependent gene
silencing by RNAi had been reported [6]. In this case,
the RNAi effect on Drosophila sex differentiation observed
at 29 C was strongly inhibited at 22 C. A
temperature-sensitive RNAi effect as an anti-viral reaction
was also reported in the plant system [7]. In
this case, the sensitive temperature was below 24 C
with disruption of the siRNA generation. The temperature
dependency of RNAi might also be highly
conserved among eukaryotes. And, we empirically
have noticed that culturing mammalian cells in a
moderate hypothermic temperature often supports the
ectopic gene expression (a confirmative experiment on
the expression of the green fluorescent protein (GFP))
in the HEK 293 and NIH-3T3 cells is indicated in
Fig. 1A). We expected that the hypothermic temperature
also suppresses the mammalian RNAi to support
the ectopic gene expression, because the
suppression of RNAi greatly enhances the ectopic gene
expression in plant system [7,8]. In this report, we
tried to analyze the effect of different temperatures, on
the efficacy of RNAi in cultured mammalian cells,
and found a suppressive effect at hypothermic
temperatures.
Discussion
In this study, we found a temperature-sensitive RNAi
effect on several mammalian cells (Figs. 1–3) as in the
insect [6] and plant [7] systems, suggesting its conservation
among eukaryotes. The temperature-sensitivity
of the RNAi effect on the expression of GFP and LacZ
genes differed (Fig. 2B). In the case of GFP, RNAi by
two different siRNAs with a different GC content
(GFP5¼52.6%, siGFP¼71.4%) was similarly and
markedly affected by the hypothermic-temperature,
while the RNAi effect on LacZ by siLacZ (GC content
¼47.4%) was relatively resistant to the hypothermic-
temperature, suggesting that the temperature and
degree of sensitivity differ among target genes (Fig. 2B).
This would be a reason for the high level expression of
ectopic gene in mammalian cells at a hypothermic temperature
(Fig. 1A) and could be experimentally and
therapeutically useful for the fine control of the effect of
RNAi on a specific target gene by controlling the temperature
in vitro and in vivo.
The biological meaning of the temperature-sensitive
RNAi in homeothermic mammals is not clear. However,
it is well known that the thermal environment affects
development, metabolism, and behavior of poikilotherms,
including sex determination and hibernation [14].
The heterochromatin formation and chromatin-based
gene silencing highly correlate with the suppression of
internal repetitive transposable elements by RNAi [15–
17]. Suggestively, the position effect variegation (PEV),
an epigenetic gene regulation by vicinal heterochromatin
[18,19], and the activation of internal latent transposons
[20,21] are temperature-sensitive in the insect, plant, and
fungi systems. To expect, analogically, that the temperature-
sensitive RNAi in poikilotherms, or also in hypothermic
mammals, could epigenetically affect the ectopic
and internal gene expressions is not so incredible.
A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi
Takashi Kameda, Kenji Ikegami, Yang Liu, Kunihiko Terada, and Toshihiro Sugiyamundefined
Department of Biochemistry, Akita University School of Medicine 1-1-1 Hondo, Akita 010-8543, Japan
Received 12 January 2004
Abstract
RNA interference (RNAi) has been attracting a great deal of attention. This pathway is highly conserved among most eukaryotes
and believed to be important for antiviral reactions and epigenetic gene regulation. Because a temperature-sensitive RNAi was
reported in both plant and insect systems, suggesting its evolutional conservation, we analyzed the effect of different temperatures on
mammalian RNAi, targeting the ectopic gene expression, and detected suppression at hypothermic temperatures. This phenomenon
could be critical and useful to control ectopic and internal gene expressions by RNAi.
2004 Elsevier Inc. All rights reserved.
Keywords: Mammalian RNAi; Temperature biology
RNAi is a nucleotide sequence-specific RNA degradation
pathway, reactive to double-stranded RNA [1–
3]. A pathway homologous to RNAi is highly conserved
among most eukaryotes. While the biochemical mechanism
of RNAi is yet to be fully revealed, this pathway
is composed of several steps, including the production
of 21–25 nt short-interfering RNA (siRNA) from
dsRNA by the RNase III-like enzyme complex (DICER),
and the sequence-specific degradation of the
target RNA by the nuclease complex (RISC). In some
organisms, amplification of the siRNA by the RNAdependent
RNA polymerase (RdRP) and intercellular
transport of the siRNA (systemic pathway) intensify the
reaction. The RNAi in eukaryotes is thought to be an
anti-viral reaction as well as an epigenetic chromosomal
regulator, involved in the suppression of internal
transposon [1–5].
Most biochemical reactions are sensitive to temperature.
In Drosophila, temperature-dependent gene
silencing by RNAi had been reported [6]. In this case,
the RNAi effect on Drosophila sex differentiation observed
at 29 C was strongly inhibited at 22 C. A
temperature-sensitive RNAi effect as an anti-viral reaction
was also reported in the plant system [7]. In
this case, the sensitive temperature was below 24 C
with disruption of the siRNA generation. The temperature
dependency of RNAi might also be highly
conserved among eukaryotes. And, we empirically
have noticed that culturing mammalian cells in a
moderate hypothermic temperature often supports the
ectopic gene expression (a confirmative experiment on
the expression of the green fluorescent protein (GFP))
in the HEK 293 and NIH-3T3 cells is indicated in
Fig. 1A). We expected that the hypothermic temperature
also suppresses the mammalian RNAi to support
the ectopic gene expression, because the
suppression of RNAi greatly enhances the ectopic gene
expression in plant system [7,8]. In this report, we
tried to analyze the effect of different temperatures, on
the efficacy of RNAi in cultured mammalian cells,
and found a suppressive effect at hypothermic
temperatures.
Discussion
In this study, we found a temperature-sensitive RNAi
effect on several mammalian cells (Figs. 1–3) as in the
insect [6] and plant [7] systems, suggesting its conservation
among eukaryotes. The temperature-sensitivity
of the RNAi effect on the expression of GFP and LacZ
genes differed (Fig. 2B). In the case of GFP, RNAi by
two different siRNAs with a different GC content
(GFP5¼52.6%, siGFP¼71.4%) was similarly and
markedly affected by the hypothermic-temperature,
while the RNAi effect on LacZ by siLacZ (GC content
¼47.4%) was relatively resistant to the hypothermic-
temperature, suggesting that the temperature and
degree of sensitivity differ among target genes (Fig. 2B).
This would be a reason for the high level expression of
ectopic gene in mammalian cells at a hypothermic temperature
(Fig. 1A) and could be experimentally and
therapeutically useful for the fine control of the effect of
RNAi on a specific target gene by controlling the temperature
in vitro and in vivo.
The biological meaning of the temperature-sensitive
RNAi in homeothermic mammals is not clear. However,
it is well known that the thermal environment affects
development, metabolism, and behavior of poikilotherms,
including sex determination and hibernation [14].
The heterochromatin formation and chromatin-based
gene silencing highly correlate with the suppression of
internal repetitive transposable elements by RNAi [15–
17]. Suggestively, the position effect variegation (PEV),
an epigenetic gene regulation by vicinal heterochromatin
[18,19], and the activation of internal latent transposons
[20,21] are temperature-sensitive in the insect, plant, and
fungi systems. To expect, analogically, that the temperature-
sensitive RNAi in poikilotherms, or also in hypothermic
mammals, could epigenetically affect the ectopic
and internal gene expressions is not so incredible.
