If the null hypothesis that the data come from a normal distribution cannot be rejected, Student's t -test was used. Otherwise, the Mann-Whitney test was used. Due to the strong non-normality of the behavior data, Mann-Whitney test was used for all the behavior analysis. For categorical data, Fisher's exact test was used.
We thank K. Sullivan for behavior annotation and scoring; R. Hellmiss for figure artwork; E. Soucy and J. Greenwood for technical assistance. We also thank members of the Dulac and Uchida laboratories and V. Murthy, A. Schier and M. Meister for advice on experiments and statistical analysis and comments on the manuscript, and the anonymous reviewers for their helpful suggestions and comments.
A performed the experiments and collected the data. National Center for Biotechnology Information , U. Author manuscript; available in PMC Nov Zheng Wu , 1 Anita E. Autry , 1 Joseph F. Anita E. Joseph F. Catherine G. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Nature. See other articles in PMC that cite the published article.
Abstract Mice display robust, stereotyped behaviors toward pups: virgin males typically attack pups, while virgin females and sexually experienced males and females display parental care. Vomeronasal control of pup-directed aggression The vomeronasal system plays an essential role in regulating sex-specific behaviors Open in a separate window. Figure 1. Neuronal activation during parenting To identify brain regions involved in parental care, we compared the brain activity patterns of virgin males versus virgin females and paternal males using induction of the immediate early gene c-fos as a read-out of neuronal activation after exposure to pups.
Figure 2. Extended Data Figure 1. Extended Data Figure 2. Figure 3. Extended Data Figure 3. Extended Data Figure 4. Figure 4. Extended Data Figure 5. Extended Data Figure 6. Figure 5. Extended Data Figure 9. Extended Data Figure 7.
Behavior raster plot of Gal::ChR2 and control virgin males with and without light illumination Each row represents a single trial lasting for 5 min or until the male attacked the pup. Extended Data Figure 8. Behavior raster plot of mated Gal::ChR2 and control males with and without light illumination Each row represents a min trial. Discussion Our data provide significant insights into the control of opposing social behaviors in mice: parenting versus pup-directed aggression.
Behavior assay Before behavior tests animals were housed individually for about one week. Parental behavior assay for mated males Fig. RNA in situ hybridization Fresh brain tissues were collected from animals housed in their home cage or 35 minutes after the start of the behavior tests when c-fos expression is analyzed.
Immunohistochemistry Immunohistochemistry was performed according to standard protocols. Statistics The sample sizes in our study were chosen based on common practice in animal behavior experiments.
Acknowledgement We thank K. Footnotes Contributions Z. Competing financial interests The authors declare no competing financial interests. References 1. Numan M, Insel TR. The Neurobiology of Parental Behavior. Springer; Sex differences in the parental behavior of rodents. Neuroscience and biobehavioral reviews. Brown R. Hormonal and experiential factors influencing parental behaviour in male rodents: an integrative approach.
Behavioural Processes. Rosenblatt J. Nonhormonal basis of maternal behavior in the rat. Terkel J, Rosenblatt JS. Maternal behavior induced by maternal blood plasma injected into virgin rats. Journal of comparative and physiological psychology.
Hormonal induction of maternal behavior in the ovariectomized nulliparous rat. Svare B, Mann M. Infanticide: Genetic, developmental and hormonal influences in mice. Brooks RJ, Schwarzkopf L. Factors affecting incidence of infanticide and discrimination of related and unrelated neonates in male Mus musculus.
Behavioral and neural biology. Labov JB. Factors influencing infanticidal behavior in wild male house mice Mus musculus. Behavioral Ecology and Sociobiology.
The regulation of infanticide and parental behavior: implications for reproductive success in male mice. Vom Saal FS. Time-contingent change in infanticide and parental behavior induced by ejaculation in male mice. Numan M, Stolzenberg DS.
Medial preoptic area interactions with dopamine neural systems in the control of the onset and maintenance of maternal behavior in rats. Frontiers in neuroendocrinology. Numan M. Medial preoptic area and maternal behavior in the female rat.
Sexual dimorphism in the vomeronasal pathway and sex differences in reproductive behaviors. Brain research. Brain research reviews. Loss of sex discrimination and male-male aggression in mice deficient for TRP2. Science New York, N. A functional circuit underlying male sexual behaviour in the female mouse brain. Mennella JA, Moltz H. Infanticide in the male rat: the role of the vomeronasal organ. Journal of Neuroscience.
Vomeronasal and olfactory system modulation of maternal behavior in the rat. TRP2: a candidate transduction channel for mammalian pheromone sensory signaling. Elwood RW. Inhibition of infanticide and onset of paternal care in male mice Mus musculus. Journal of Comparative Psychology. Calamandrei G, Keverne EB. Differential expression of Fos protein in the brain of female mice dependent on pup sensory cues and maternal experience. Behavioral neuroscience.
In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon TC.
To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs. These PTCs could originate from mutations as well as transcriptional or splicing errors 1. The fact that NMD targets endogenous mRNAs that encode functional proteins revealed an important role of NMD as a post-transcriptional gene expression regulation mechanism, affecting a series of biological functions ranging from tissue differentiation to protection of host cells from RNA viruses reviewed in refs.
Since NMD is crucial for a wide range of biological functions, an accurate and highly specific recognition mechanism of mRNAs that need to be engaged in the pathway is essential. An important feature of NMD is that it is dependent on translation, either on the first or a later round 9 , 10 , 11 , Evidence supporting the formation of complexes containing NMD and translation termination factors suggested a functional connection between the two processes 13 , 14 , 15 , 16 , In view of this data, a currently prevailing working model for NMD activation suggests that NMD ensues when translation termination is aberrant, either because it requires additional factors for ribosome release or because it is not fast enough.
Based on evidence from S. These data entertain the hypothesis that the difference between productive and aberrant, NMD-eliciting translation termination might rely on whether PABPC1 or UPF1 interacts with eRF3 at the terminating ribosome, thereby either promoting or inhibiting translation termination It should be noted, however, that PABP has been shown to stimulate translation termination only under non-physiological, limiting concentrations of release factors using a eukaryotic reconstituted system Using a similar approach, it was suggested that under decreased concentrations of release factors, the conserved NMD factor UPF3B can delay translation termination and dissociate post-termination ribosomal complexes that are devoid of a nascent peptide Previous approaches in measuring ribosomal density at the TC of reconstituted mammalian in vitro translation systems have offered important insights into the roles of crucial translation termination factors 26 , However, the fact that they are developed based on short open reading frames and that the reconstituted systems lack essential NMD factors limits their application in understanding translation termination in the context of NMD.
Here we present the development of an in vitro biochemical approach that allows the detection and comparison of ribosomal density at the TC using human cell lysates and in vitro transcribed mRNAs. We show that in this system, the occupancy of ribosomes at the TC is similar on both NMD-sensitive and insensitive reporter mRNAs, as well as in the presence or absence of a poly A tail.
We complement these results by comparing the ribosomal density at the TC of mRNAs in vivo by ribosome profiling, which also revealed a similar occupancy of ribosomes at TCs, independently of their sensitivity to NMD.
Previous work has suggested that prolonged ribosomal occupancy at the TC is a characteristic feature and maybe even the trigger for an mRNA to be subjected to the NMD pathway 18 , In order to assess ribosomal density at the TC, we developed an in vitro assay that allowed us to reproducibly identify terminating ribosomes on in vitro transcribed reporter mRNAs, based on previous protocols for the production of translation-competent cell lysates We opted for an approach that would allow all stages of translation of different mRNAs to occur using HeLa cell lysates followed by toeprinting assays.
After optimization of different parameters such as mRNA concentration, incubation time, and titration of magnesium concentration to ensure efficient translation activity Supplementary Fig. No luminescence, and hence translation activity, was detected in the presence of the translation inhibitor puromycin.
After purification, the cDNA molecules were analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. A puromycin-treated control reaction was used to distinguish between translation-dependent toeprints and translation-independent break-offs of the reverse transcriptase. To observe reproducible translation-dependent toeprints originating from ribosomes at TCs, we titrated a broad range of biochemical parameters. This technical improvement allowed us, for example, to produce lysates from cells with an siRNA-mediated knockdown.
As shown in Fig. To distinguish whether translation-dependent bands correspond to ribosomes preventing the reverse transcriptase from passing or whether they originate from mRNA molecules cleaved during translation, we phenol-extracted the mRNA molecules prior to the primer extension step.
While such cleaved mRNA fragments were detected in a series of toeprint experiments performed in rabbit reticulocyte lysates Supplementary Fig. Moving the TC 6 or 15 nucleotides downstream of the original position resulted in a corresponding shift of the translation-dependent bands, confirming that our assay reliably detects toeprints originating from ribosomes at stop codons Fig.
The red arrow denotes the radiolabeled toeprint primer binding 66 nts downstream of the TC orange dot. To locate the positions of the toeprints, a Sanger sequencing reaction was run in parallel G, T, C, A using the same primer.
The translation-dependent bands corresponding to toeprints from ribosomes at the respective TCs are marked with dots following color code of d. In vitro translation and toeprint analysis were performed as in c. Source data are provided as a Source Data File. The second reporter transcript served as a control, as it does not lead to ribosome stalling. The two reporter mRNAs were translated in HeLa cell lysates and were subjected to toeprint analysis as described before.
Overall, the above results verified that our system can reproducibly detect terminating ribosomes as well as differences in ribosomal density at the termination codon. The red arrow denotes the radiolabeled toeprint primer and the orange dot the termination codon.
Sanger sequencing reactions were run in parallel G, T, C, A to locate the positions of the toeprints. The band corresponding to full-length Rluc protein is denoted by an arrow. The experiment was repeated three times. Next, we wanted to assess whether our toeprint assay is also able to identify changes of ribosome density at the TC under aberrant translation termination conditions.
A previous study showed that reduction of the eukaryotic recycling factor ABCE1 can lead to prolonged ribosome occupancy at the TC The efficacy of the ABCE1 knockdown was documented by probing equal amounts of lysates by western blotting Fig.
This confirms that our in vitro system has the capacity to monitor differences in ribosome occupancy at the TC after depleting translation termination-related factors. The reporters were designed in a way that allows the comparison of ribosome occupancy at the TC in toeprint assays using the same radiolabeled primer.
The efficacy of the UPF1 knockdown was monitored by western blotting Fig. The constructs were cloned into pcDNA3. It has been proposed that a long physical distance between the TC and the poly A binding protein PABP may hinder translation termination by causing prolonged ribosomal occupancy at the TC 18 , Previous reports have also suggested that PABP can facilitate translation termination under decreased release factors concentrations in vitro 23 and we therefore wanted to assess in our toeprint assay whether ribosome occupancy at the TC is affected by the presence or absence of a poly A tail, or by its physical distance from the TC.
To this end, equimolar amounts of the in vitro transcribed, capped and purified reporter mRNAs were used for translation in HeLa lysates as described above, followed by primer extension reactions. However, it is also possible that our in vitro toeprinting assay may simply not fully recapitulate the situation in vivo. TC: orange dot. Sanger sequencing reactions were run in parallel G,T,C,A. Transcripts were aligned to the stop codon 0 and the mapped reads from nucleotides upstream to nucleotides downstream of the stop codon are shown.
Transcripts were ordered according to their ribosome occupancy at the termination codon transcripts with the highest number of reads at the stop codon at the top. NMD-sensitive transcripts are shown on the left of the heatmap panels. Each pixel line corresponds to the average of 10 transcripts.
Violin plots showing the counts distribution of ribosome-derived reads mapping at the stop codon relative to ribosome-derived reads aligning to the CDS as an average of 3 biological replicates.
Boxplots middle indicate the percentiles 5,25,50,75, and 95 of the ribosome occupancy values distribution with dots representing outliers. Percentage of reads relative to total reads are shown on the y-axis. The ribosome profiling protocol was modified to assess specifically ribosome occupancy at the TC of endogenous mRNAs by omitting cycloheximide and applying instead snap-freezing of the cell lysates in liquid nitrogen Metagene analysis of ribosome-associated footprints from three independent experiments showed that under these conditions, ribosomes tend to reside longer on average at the TC than at a codon within the ORF Fig.
To normalize for the overall translation efficiency of a given mRNA, we determined the relative ribosome occupancy at TCs compared to CDS for each transcript by defining the ratio of the total counts of ribosome-derived reads aligning to the TC relative to the average of ribosome reads mapping at the CDS. In addition, omitting a poly A tail from our reporter constructs did not affect the ribosome occupancy at the TC.
These results from our in vitro toeprinting system are corroborated by our ribosome profiling experiments, in which normalized ribosome occupancy at TCs of mRNAs did not correlate with whether an mRNA was a target for the NMD pathway or not. Thus, our in vitro and in vivo data are in disagreement with a suggested working model for NMD, which posits that inefficient or aberrant translation termination that can be detected as ribosome stalling at the TC is the signal for NMD to ensue 18 , In vitro translation systems have significantly contributed to assess a broad range of translation-related mechanisms 39 , 40 , However, the study of translation termination in vitro using mammalian systems has remained technically challenging and has only recently gained broader attention.
The cryo-electron microscopic analysis of ribosomal complexes isolated from in vitro translation reactions in rabbit reticulocyte lysate yielded structural information about distinct steps of translation 42 , and fully reconstituted eukaryotic systems allowed a detailed, stepwise functional interrogation of translation termination complexes and of the roles of release factors 26 , In this context, reconstituted translation is performed with isolated ribosomes that are supplemented with purified translation factors and aminoacylated tRNAs.
The separate addition of each component allows tight control of the system and its modulation e. On the other side, translation factor concentrations are far from physiological conditions and there is a requirement of intermediate purification steps of ribosome complexes, which limits the biological relevance of reconstituted translation systems.
While they are well suited to study mechanistic aspects of translation, they will fail to recapitulate many steps of translation regulation and processes more accessorily connected to translation. Since we are mainly interested in investigating the connection between translation and NMD, we opted for the development of a more physiologically relevant human-based in vitro system that does not require the isolation of specific ribosomal sub-complexes.
This system allows all steps of translation of the in vitro transcribed reporter mRNA in the lysates to occur before toeprinting and detection of ribosomes at the TC.
The system can be modulated, for example by knocking down specific factors or by the addition of recombinant proteins. We based our in vitro system on HeLa cells, because the majority of biochemical data available concerning human NMD derives from studies in this system.
The development of an assay that allows monitoring of ribosome occupancy at the TC was an important achievement to test the idea that prolonged ribosome stalling at the TC is indeed the trigger for NMD. Our in vitro toeprinting approach allows a direct comparison of ribosome occupancy at TCs between different reporter mRNAs translated in lysates of human cells. Extensive titration and optimization of different biochemical parameters Supplementary Fig.
Optimization of our system was focused on yielding reproducible translation-dependent bands in toeprint assays Fig. Toeprint assays under the established conditions allow the reproducible detection of ribosomes at TCs and changes in the ribosome occupancy induced by stalling peptide sequences Fig. The fact that our assay can detect ribosomes at the TC rather than at codons further upstream is in agreement with our ribosome profiling data that also showed an overall higher occupancy of ribosomes at the TC compared to the ORF.
These two results agree with the notion that translational pauses at the termination codon is a common feature of translation As aforementioned, comparison between NMD-sensitive and NMD-insensitive mRNA reporters revealed a similar occupancy of ribosomes at the TC both in our in vitro toeprinting assay as well as by in vivo ribosome profiling Fig.
This data contrasts with two previous reports according to which increased ribosome density was observed on PTC-containing transcripts in yeast extracts and in rabbit reticulocyte lysates 18 , While it is conceivable that the difference between the yeast system and the human system reflects indeed a species difference, we speculate that the difference between the rabbit reticulocyte lysate and the human system might be attributed to toeprints originating from RNA cleavage in the reticulocyte lysate.
We had initially also developed our toeprint assay using rabbit reticulocyte lysate and found that the toeprints we detected in this system almost exclusively represented co-translational RNA cleavages next to the ribosome Supplementary Fig. For this reason, we performed an additional control to monitor whether toeprints originate from ribosomes or RNA cleavage events by re-purifying the RNA after translation and prior to primer extension.
A side-by-side comparison between these phenol-purified and untreated samples reveals whether toeprints derive from ribosomes or from cleaved RNAs.
The extent of increased ribosome occupancy at the termination codon may vary and stimulate different effects on mRNAs that are monitored by translation-dependent pathways. In cases of faulty mRNAs that are degraded due to a lack of a TC nonstop decay or due to a strong secondary structure or the absence of a cognate tRNA no-go decay , the activating signal is the ribosome stalling during elongation.
In this scenario, ribosome stalling marks a dead-end event and the rescue of an otherwise trapped ribosome is crucial reviewed in refs. Even though a clear distinction between the two is still technically difficult, stalling as well as ribosome pausing have clearly been detectable by means of ribosome profiling or toeprinting 46 , 47 , 48 , 49 and both could be reproduced in our in vitro system. While we cannot exclude the possibility that NMD is associated with transient delays of translation at the termination codon that are too brief to be captured by means of toeprinting or ribosome profiling, our data clearly argue against the occurrence of stable ribosome stalling at TCs of NMD-sensitive mRNAs.
There is a growing body of evidence that NMD occurs stochastically depending on highly variable interactions that finally lead to the degradation of the mRNA, opposed to the traditional view of a linear, ordered, and irreversible pathway that leads to decay 7. In yeast, NMD substrates were found to have an increased rate of out-of-frame translation, accompanied by an overall decreased codon optimality or stretches of non-optimal codons compared to NMD-insensitive mRNAs For example, we may use these cookies to determine if you have interacted with a certain page.
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