C. elegans, have successfully identified genes required to process natural sensory stimuli. However, functional redundancy within the underlying neural circuits, which are often organized with multiple parallel neural pathways, limits our ability to identify ‘neural pathway-specific genes’, i.e. genes that are essential for the function of some, but not all of these redundant neural pathways. To overcome this limitation, we developed a ‘forward optogenetics’ screening strategy in which natural stimuli are initially replaced by the selective optogenetic activation of a specific neural pathway. We used this strategy to address the function of the polymodal FLP nociceptors mediating avoidance of noxious thermal and mechanical stimuli. According to our expectations, we identified both mutations in ‘general’ avoidance genes that broadly impact avoidance responses to a variety of natural noxious stimuli  and mutations that produce a narrower impact, more restricted to the FLP pathway . Through a detailed follow-up analysis, we further showed that the Ryanodine receptor UNC-68 acts cell-autonomously in FLP to adjust heat-evoked calcium signals and aversive behaviors. As a whole, our work (i) reveals the importance of properly regulated ER calcium release for FLP function, (ii) provides new entry points for new nociception research and (iii) demonstrates the utility of our forward optogenetic strategy, which can easily be transposed to analyze other neural pathways.Understanding how the nervous system bridges sensation and behavior requires the elucidation of complex neural and molecular networks. Forward genetic approaches, such as screens conducted in  Caenorhabditis elegans model that overcomes this intrinsic robustness and enables the recovery of mutations with a non-pervasive impact, but nevertheless important for the function of specific neural pathways. After mutation mapping and through a series of additional experiments, we could identify novel avoidance genes and highlight intracellular signaling mechanisms working in specific nociceptor neurons. Our approach can be transposed to analyze other neural pathways and bring a more comprehensive view of the molecular network that is used by the nervous system to bridge sensation and behavior.One of the main functions of the nervous system is to process sensory information about the environment and produce useful behaviors. For example, potentially damaging stimuli are detected by specialized neurons called nociceptors and sensory information processed to trigger avoidance behaviors. The nociceptive functions are largely conserved from animals to human, but the underpinning molecular mechanisms are far from being completely understood. Mutating genes in model organisms and screening for impaired avoidance is a powerful way to identify new molecular factors in nociceptive pathways. However, the approach is limited by the robust nervous system architecture often presenting parallel pathways with backup functions. Here, we introduce an experimental approach in the  C. elegans is an ideal model for addressing the molecular, cellular and circuit basis of behavior. The analysis of C. elegans sensory behavior neural circuits revealed in many instances a high level of degeneracy animals were grown in the presence of the all-trans-retinal (ATR) co-factor, but failed to do so in the absence of ATR . Third, we adjusted the number of stimuli delivered to each animal and the criterion for retaining a candidate mutant. Because FLPs are glutamatergic neurons, we choose an eat-4 mutant, known to selectively affect the loading of vesicles with glutamate  transgenic animals expressing unc-4 cDNA under the control of a 2 kb unc-4 promoter region (unc-4(dom9) is a loss-of-function allele and that UNC-4 is required for the FLP-evoked reversal response, which is consistent with a general impairment in backward locomotion transgenic animals expressing unc-14 cDNA under the control of a 2 kb unc-14 promoter region, confirming that intact UNC-14 is necessary for this behavior (unc-14(dom8) animals tended to coil, as described for other unc-14 loss-of-function mutants  transgenic animals expressing syd-2 cDNA under the control of a 2 kb syd-2 promoter region  mutation was the cause of the FLP-mediated avoidance defect, we engineered the same mutation with CRISPR/Cas9 genome editing. We observed a similar phenotype in the resulting unc-68(syb212) mutant (unc-68(dom13) was partially rescued in transgenic animal lines made with a fosmid covering the whole unc-68 gene region. Control transgenic animals made with a cosmid that did not cover the full unc-68 coding sequence failed to rescue unc-68(dom13). These results establish that the unc-68(dom13) mutation is indeed responsible for the FLP-evoked reversal response defect.VDM placed the n 6–9 Mb . This re protein . RyRs ar elegans . To veri) mutant . Furtherunc-68(dom13) is a loss- or a gain-of-function mutation. The semi-dominant behavior of this allele (unc-68(dom13) is indeed a gain-of-function mutation. First, we compared the impact of unc-68(dom13) with that of recessive null mutations previously identified: unc-68(r1161) and unc-68(r1162). Whereas homozygote mutants for either null allele displayed a significant reduction in the FLP-evoked avoidance behavior, this effect was significantly less pronounced than that in unc-68(dom13) mutants (unc-68(r1161) allele and one copy of the unc-68(dom13) allele. If the dom13 allele was a loss-of-function, one would expect to see a similar or less dramatic phenotype than that in null homozygotes. Conversely, if it was a gain-of function allele, the phenotype should be more severe than in the null homozygotes. Our results are in agreement with the latter model, with unc-68(r1161/dom13) having a stronger reduction in FLP-evoked avoidance than the null  was encoding an over-activated channel, we found that this mutant had a dramatic swimming deficit, more pronounced than that in the unc-68(r1161) null  animals had a reduced pharyngeal pumping rate (unc-68(dom13)  null . Fourth,ly shown , unc-68(ing rate . The ave8(dom13) , but forribution  and an iribution .unc-68(dom13) is a gain-of-function mutation, most likely encoding a deregulated channel. Furthermore, since both gain- and loss-of-function mutations in unc-68 impair nociceptive behavioral responses, these results also suggest that an appropriate level of UNC-68 activity is essential for FLP-evoked avoidance.Collectively, these results suggest that The mutations identified in our screen impair avoidance responses triggered by an optogenetic activation of FLP, which is an artificial situation. We next addressed whether these mutations could also impact the animal response to natural stimuli, starting with stimuli known to be detected by FLP.unc-4(dom9), as well as in unc-4(e120), another loss-of-function allele. Conversely, the sensitivity to head poking in eat-4, unc-14, unc-83, syd-2 and unc-68 mutants was unaffected, which is in striking contrast with the strong impairment in optogenetically evoked FLP-specific responses . In forward-moving wild type adults, head poking induced robust reversal responses . Consistesponses . These runc-4 mutants, an effect even stronger than that observed in a double osm-9 ocr-2 mutant used as positive control in our assays  transgene, but left unaltered by the [mec-3p::unc-4(cds)]transgene  mutation was equally and markedly rescued by a eat-4-carrying fosmid, the [eat-4p::eat-4(cds)] transgene, and the [mec-3p::eat-4(cds)] transgene  and its coding sequence (>15 kb), we had to engage a different set of transgenic tools than the one used in the previous section for unc-68 null mutants could be rescued by tissue-specific expression of wild type UNC-68. We examined the effect of muscle-specific and pan-neuronal rescue by using previously generated transgenic lines, in which the large unc-68 gene region contained in a cosmid had been recombined in vivo to be controlled by myo-3 or rab-3 promoters, respectively ([unc-68(r1161) mutation  null animals ([QUAS::unc-68(frameshift)] fosmid did not produced any rescue effect. With this functional rescue system, we next combined the [QUAS::unc-68] fosmid with a [mec-3p::QF::SL2::mCherry] plasmid that drives robust expression in FLP and in the tail PLM neurons, and more sporadic expression in a few other neurons. This construct combination produced a partial rescue of FLP-evoked reversals, similar to that obtained with the unc-68 promoter  null, suggesting a partial loss of function with RNAi. No effect was found in controls where only the sense RNA was expressed. We obtained similar results when inducing RNAi with the mec-3 promoter. Although the implication of other neurons is not excluded, our results suggest that UNC-68 expression in FLP is required for FLP-evoked avoidance.Third, we examined if a cell-specific knock-down of equences . Inducinvoidance . This efunc-68 mutations on the FLP response to noxious heat stimuli, in a line expressing a Cameleon calcium indicator in FLP  transgene were treated with 50mM ethyl metanesulphonate (EMS) mutagen for 4h in M9 solution in a 15 mL Falcon tube submitted to continuous rotation, as previously described  animals expressing the Cameleon sensor in their FLP neurons were pre-selected by using an epi-fluorescence stereomicroscope at least one hour prior to the experiment. For each recording, one worm was collected with an eyelash pick, transferred to an agarose pad, and then immobilized by applying Dermabond surgical glue , using glass capillary needles operated by mouth. The glue was initially applied close to the tail of the worm, on its dorsal side  and then along the whole body, until close to the head but without gluing the nose. After glue polymerization (around 30 s), M9 buffer was added close to the worm to keep the environment slightly humid and prevent exsiccation of the agarose pads. A plastic spacer was placed on the coverslip between the worm and a Cherry Temp microfluidic chip . Sample temperature could be stably maintained (variations < 0.1°C) or quickly changed to a new steady temperature (within 10 s), thanks to a circuit switch between two pre-equilibrated Peltier elements located near the sample. The temperature of the microfluidic chamber was preset to 20°C to maintain a constant baseline temperature before initializing the experimental protocols. Stimulation was at 36°C for 30 s.0.3 mm-thick agarose pads were prepared with 2% agarose in M9 buffer (KHdescribed. TransgeWorms were imaged using an inverted epifluorescence microscope (Leica DMI6000B) with aHCX PL Fluotar L40x/0.60 CORR dry objective, a Leica DFC360FX CCD camera , an EL6000 Light Source, and fast filter wheels for FRET imaging. Excitation filters for CFP and FRET: 427 nm (BP 427/10). Emission filters for CFP: 472 (BP 472/30) and for FRET: 542 nm (BP 542/27). Dichroic mirror: RCY 440/520. Five minutes of initial recording at 20°C was used as baseline. The acquisition rate was 1Hz.background values were subtracted from the ROIFLP values. The resulting values for FRET and CFP respectively (namely FRETFLP-background and CFPFLP-background) were divided and the FRET ratio (FRET/CFP) was calculated as follows:All time-lapse recordings were analyzed using LAS X software (Leica). To calculate FRET ratio, we selected a first region of interest (ROI), delimiting the background, and a second ROI, which included FLP soma. For each channel, the ROI R was normalized and expressed as a ΔR/R0 at every time point, where ΔR is R-R0 and R0 is the average of R over the last 30 s prior to stimulus onset. To compare baseline intracellular calcium levels between genotypes, we first verified that the Cameleon sensor was expressed at similar levels between them. To do so, we computed a total fluorescence index (FRETFLP-background + CFPFLP-background) as an indicator of the Cameleon sensor expression level, and found it was not significantly different between genotypes . Then, we compared the baseline FRET ratio 0R between genotypes  was microinjected in the gonad to generate transgenic lines according to a standard protocol . We used animals .[eat-4 fosmid], M04C11 [unc-68 cosmid], and W01A11 [unc-83 cosmid] were obtained from the Wellcome Sanger Institute .F23K23 [unc-68 fosmid] was obtained from Source BioScience. The generation of the [QUAS::unc-68] fosmids is described below.WRM069cA02 Entry plasmids containing specific promoters were constructed by PCR from N2 genomic DNA with primers flanked with attB4 and attB1r recombination sites and cloned into pDONR-P4-P1R vector (Invitrogen) by BP recombination. Primer sequences were the following:dg596[slot1 Entry unc-14p]attB4unc-14_F: ggggacaactttgtatagaaaagttgatcaaagagagaggtggaaactgaacaattB1runc-14_R: ggggactgcttttttgtacaaacttgtttgagggatgaagccctttgtagdg598[slot1 Entry syd-2p]attB4syd-2_F: ggggacaactttgtatagaaaagttgatagtgagtttgactggctctactattB1rsyd-2_R: ggggactgcttttttgtacaaacttgtaccttgcagaaagtgaataacctacadg600[slot1 Entry unc-4p]attB4unc-4_F: ggggacaactttgtatagaaaagttgattatgaatgctgtcccggaactgattB1runc-4_R: ggggactgcttttttgtacaaacttgttttcactttttggaagaagaagatcctctdg712[slot1 Entry eat-4p]attB4eat-4_F: ggggacaactttgtatagaaaagttgatgtgccatccgtctatttccagaatgaattB1reat-4_R: ggggactgcttttttgtacaaacttgtctgaaaatgatgatgatgatgatggagttgdg604[slot1 Entry unc-68p]attB4unc-68AB_F: ggggacaactttgtatagaaaagttgatcgttggttaataattgttggctaaccgtattB1runc-68AB_R: ggggactgcttttttgtacaaacttgtctgtaaaacaaaaaaactagaggtgctggdg672[slot1 Entry rab-3p1208]attB4rab-3p1208_F: ggggacaactttgtatagaaaagttgatgatcttcagatgggagcagtggactattB1rrab-3p1208_R: ggggactgcttttttgtacaaacttgtagccgccatctgaaaatagggctadg68[slot1 Entry mec-3p_noATG] and dg229[slot1 Entry QUASprom] was previously described [The generation of escribed .Entry plasmids containing specific coding DNA sequences (cds) were constructed by PCR from N2 cDNA with primers flanked with attB1 and attB2 recombination sites and cloned into pDONR_221 vector (Invitrogen) by BP recombination. Primer sequences were the following:dg595[slot2 Entry unc-14cds]attB1unc-14_F: ggggacaagtttgtacaaaaaagcaggcttaatggtggaactgtgcgaactgcaattB2unc-14_R: ggggaccactttgtacaagaaagctgggttcaaactcggttgaattgggtggcatdg597[slot2 Entry syd-2cds]attB1syd-2_F: ggggacaagtttgtacaaaaaagcaggcttaatgagctacagcaatggaaacataaattB2syd-2_R: ggggaccactttgtacaagaaagctgggtttcgtttagactagcacgaatgadg599[slot2 Entry unc-4cds]attB1unc-4_F: ggggacaagtttgtacaaaaaagcaggcttaatgatcggtgcactgcatgcatattB2unc-4_R: ggggaccactttgtacaagaaagctgggttaaataaccaatggagggcacggttcdg713[slot2 Entry eat-4cds]attB1eat-4_F: ggggacaagtttgtacaaaaaagcaggcttaatgtcgtcatggaacgaggctattB2eat-4_R: ggggaccactttgtacaagaaagctgggttgtagttctggtttttcgcaactaggcdg621[slot2 Entry unc-68 RNAi sense]attB1unc-68RNAis_F: ggggacaagtttgtacaaaaaagcaggcttactgagagcagttcgattaggtccaaattB2unc-68RNAis_R: ggggaccactttgtacaagaaagctgggttctgaaggacctccttcggaaattgtdg622[slot2 Entry unc-68 RNAi antisense]attB1unc-68RNAias_F: ggggacaagtttgtacaaaaaagcaggcttatctgaaggacctccttcggaaattgtattB2unc-68RNAias_R: ggggaccactttgtacaagaaagctgggttctgagagcagttcgattaggtccaadg240[slot2 Entry QF_withATG] was previously described [The generation of escribed .dg651[slot2 Entry egl-13NLS::wrmScarlet], we first obtained a codon-optimized wrmScarlet version containing three artificial introns via gene synthesis (Eurofins DNA), amplified it by PCR to subclone it into pDON221 by BP recombination (creating dg642), and added the egl-13NLS by PCR. We used the following primers:To create NLSRightHalf_wrmScarlet_F: AAAACGCGAAGAAGCTTGCCAAGGAAGTTGAAAATGGATCCATGGTCAGCAAGGNLSLeftHalf_Entry_R: CACTCAGTTTTGTCGGATTCGCTTTTCGTCTACGGCTCATGTTGCTAGCGGTACCTAAGmg277[SL2::mCherry] was previously described [escribed .mg211[EntrySlot3unc-54UTR] (aka pMH473) was a gift from Marc Hammarlund.dg396[coel::GFP] (or unc-122p:GFP) was a gift from Piali Sengupta (Addgene plasmid # 8937)dg243[mec-3p::QF::unc-54UTR] was previously created through a LR recombination reaction between dg68, dg240, mg211, and pDEST-R4-P3.dg571[unc-4p::unc-4cds::SL2::mCherry] was created through a LR recombination reaction between dg600, dg599, mg277, and pDEST-R4-P3.dg715[eat-4p::eat-4cds::SL2::mCherry] was created through a LR recombination reaction between dg712, dg713, mg277, and pDEST-R4-P3.dg572[unc-14p::unc-14cds::SL2::mCherry] was created through a LR recombination reaction between dg596, dg595, mg277, and pDEST-R4-P3.dg574[syd-2p::syd-2cds::SL2::mCherry] was created through a LR recombination reaction between dg598, dg597, mg277, and pDEST-R4-P3.dg717[mec-3p::unc-4cds::SL2::mCherry] was created through a LR recombination reaction between dg68, dg599, mg277, and pDEST-R4-P3.dg716[mec-3p::eat-4cds::SL2::mCherry] was created through a LR recombination reaction between dg68, dg713, mg277, and pDEST-R4-P3.dg709[mec-3p::unc-14cds::SL2::mCherry] was created through a LR recombination reaction between dg68, dg595, mg277, and pDEST-R4-P3.dg710[mec-3p::syd-2cds::SL2::mCherry] was created through a LR recombination reaction between dg68, dg597, mg277, and pDEST-R4-P3.dg628[mec-3p::unc-68senseRNAi::SL2::mCherry] was created through a LR recombination reaction between dg68, dg621, mg277, and pDEST-R4-P3.dg629[unc-68pAB::unc-68senseRNAi::SL2::mCherry] was created through a LR recombination reaction between dg604, dg621, mg277, and pDEST-R4-P3.dg632[mec-3p::unc-68antisenseRNAi::SL2::mCherry] was created through a LR recombination reaction between dg68, dg622, mg277, and pDEST-R4-P3.dg634[unc-68p::unc-68antisenseRNAi::SL2::mCherry] was created through a LR recombination reaction between dg604, dg622, mg277 and pDEST-R4-P3.dg648[unc-68pAB::QF::unc-54UTR] was created through a LR recombination reaction between dg604, dg240, mg211, and pDEST-R4-P3.dg673[rab-3p1208::egl-13NLS::wrmScarlet::unc-54UTR] was created through a LR recombination reaction between dg672, dg651, mg211, and pDEST-R4-P3.dg639[QUAS::unc-68(wt)] and dg631 rescue fosmids were obtained by fosmid recombineering, using the screening strategy of kanamycin resistance gene (kanR) insertion as previously described [[KaQU68(fs)] construct fusing: (i) a 100 base homology stretch of the pCC1FOS fosmid backbone, (ii) kanR, (iii) the QUAS promoter sequence and (iv) the first one hundred nucleotides of the unc-68 gene (with a frameshift in exon1). A [KaQU68(wt)] version was then obtained by site directed mutagenesis. Then we amplified each construct by PCR with the following primers:escribed . We firsKaQU68_F1: cacaggaaacagctatgaccatgattKaQU68_R1: gtcggccatcgttgtcgatin vivo in E. coli with the unc-68 containing WRM069cA02 fosmid, according to the protocol developed by Tursun and collaborators  signal in FLP and motor neurons. FLP nuclei were localized thanks to the mNeonGreen signal. Acquisition for quantification was made with a RFP filter cube to selectively quantify the wrmScarlet signal. Note the interrupted vertical axis. Grey bars indicate averages, with SEM as error bars. *, p < .001 by Student’s t-test. Data are pooled from two independent transgenic lines showing similar results.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


A crucial part of dynamic motions is the interaction with other objects or the environment. Floating base robots have yet to perform these motions repeatably and reliably. Force torque sensors are able to provide the full description of a contact. Despite that, their use beyond a simple threshold logic is not widespread in floating base robots. Force torque sensors might change performance when mounted, which is why in situ calibration methods can improve the performance of robots by ensuring better force torque measurements. The Model-Based in situ calibration method with temperature compensation has shown promising results in improving FT sensor measurements. There are two main goals for this paper. The first is to facilitate the use and understanding of the method by providing guidelines that show their usefulness through experimental results. Then the impact of having better FT measurements with no temperature drift are demonstrated by proving that the offset estimated with this method is still useful days and even a month from the time of estimation. The effect of this is showcased by comparing the sensor response with different offsets simultaneously during real robot experiments. Furthermore, quantitative results of the improvement in dynamic behaviors due to the in situ calibration are shown. Finally, we show how using better FT measurements as feedback in low and high level controllers can impact the performance of floating base robots during dynamic motions. Experiments were performed on the floating base robot iCub. Robots are expected to perform highly dynamical motions. Being able to perform these motions repeatably and reliably is an active research topic. A crucial part of these motions is the interaction of the robot with other objects or the environment. Whenever an interaction happens there exist an exchange of forces. Therefore, knowledge of the forces exchanged at contacts is a fundamental part of endowing robots with the ability to perform dynamical motions. The six-axis force-torque (FT) sensors convey a complete information of a contact force by providing measurements of the three axes of forces and three axes of torques. Robots with their base fixed to the ground have been using FT sensors to measure contact forces since a long time ,2,3. DesThe most common phenomena used in FT sensors to measure forces is the change in resistance of silicon due to strain . In morea priori. Removing the offset after the sensor has established contact would make the value measured by the FT sensor incorrectly. Therefore, minimizing the effect of drift in the FT sensors can improve the reliability of the sensor in floating base robots.Typically, the offset in FT sensors is removed before use. Commonly floating base robots use this sensor to detect and measure contacts. Thus, the time of collision is unknown The typical calibration procedure considers, first, identifying the offset when no load is applied on the sensor. Then, carefully placing some weights in specific positions to have well known forces and torques in order to span the space of the sensor. To resolve the coupling effects, it is necessary to have calibration points with as many orientations of the force vector as possible based on the sensor’s coordinate system. The calibration data should ideally be a representative dataset of what the sensor will be subjected to.Due to the nature of the technology used, solving with least squares remains the most popular . CalibraThe most common calibration procedure is a quasi-static calibration of the sensor. Calibration procedures can be classified into ex situ when the sensor is calibrated in a place different where is used, and in situ if it is calibrated on the place it will be used.Ex situ calibration is usually performed using specialized structures ,21,22,23There are two main contributions of this paper. The first is to provide useful tips to better exploit the Model-Based in situ calibration method with temperature compensation in a simple way. The other is to showcase the impact of the improved measurements obtained with this calibration in the dynamic performance of floating base robots.The paper is structured as follows: in In this section, the developed in situ calibration method is described in detail. Starting from the general mathematical model of the FT sensor to the problem statement that is solved through the Model-Based in situ calibration method.f is the force value in k is a constant of the material A R=Ro and The minimum of a quadratic form take place when the derivative is equal to 0. Using vector differentiation properties, the solution to Equation  can be wThe form of the solution is obtain following these steps:C and the offset o, but also When considering other phenomena as linear variables the final form of the problem can be expressed as:R with the added linear value R each column has all the raw measurements of a given raw signal.Augment the raw measurements matrix Workbench matrix by adding the coefficients regarding the added linear variable Augment the Workbench coefficients of the added linear variable a values in the diagonal or left (BSL) depending on the support leg.Two types of datasets that were used previously to calibrate a sensor.A calibration dataset could be formed by one of these kinds of dataset or a combination of them.centralized offset removal with or without temperature, using the same Previous results showed that the sphere estimation type using the Grid dataset gave the best results when no temperature was taken into account . Adding In those tests, no temperature offset was considered. The validation datasets were collected the same day in between the calibration datasets. The results shown were about the external force estimation, but how this affects robot performance was not presented.iCub Facility at the Italian Institute of Technology.The experimental platform is the floating base robot iCub. It is a child-sized humanoid robot originally developed during the RobotCub European Project for research in embodied cognition  by the iIt has 53 degrees of freedom (DoF), weighs around 33 kg and is 104 cm tall. The DoFs are distributed as in the following way: six for each leg, three for the torso, six for the head and eyes, seven for each arm and nine for each hand. One additional servo motor is used to open and close the eyelids. For the calibration, only a subset of 32 DOFs  are used. The version of iCub used is known as 2.5. It has Brushless Direct Current electric motor (BLDC) with an Harmonic Drive transmission, making them suitable for joint torque control.More details on the actuation and mechanics of the iCub 2.5 can be found in . An imagThe iCub has various sensors including inertial measurement units (IMU), force-torque (FT) sensors, cameras, microphones, joint encoders and tactile sensor arrays, that cover the surface of the robot. Six custom-made six-axis FT sensors ) are plaThe interface to interact with the iCub is through Yet Another Robot Platform (YARP). More specifically, YARP supports building a robot control system as a collection of programs communicating in a peer-to-peer way, with an extensible family of connection types  that can be swapped in and out.To estimate the FT sensors offset before experiments.As a threshold to know if a stable contact has been established between the robot and the ground.To give the feedback to the low-level joint torque controller.To estimate dynamical quantities used by high-level controllers, such as the center of pressure (CoP) and the zero moment point (ZMP).To give feedback to high-level controllers.The force-torque sensing has five main uses in the iCub:To Estimate the FT Sensors Offset before ExperimentsThe robot is hanging from the torso and mass is measured with IMU.The robot is standing on one leg and the mass is measured using IMU.The robot is standing on one leg and the mass is imposed to be acting in the axis normal to the ground.A typical sequence before using the robot, involves removing the offset when only one contact with the environment exists. This offset estimation requires the information from the mass vector. This can be imposed in a known robot configuration or measured using an IMU. This offset is then subtracted from the measurements. There are three possibilities to estimate the offset:As a ThresholdThe simplest use is as a threshold. When the load on the sensors reaches a chosen value, the controllers change state assuming a stable contact has been achieved. The chosen value can be a percentage of the total body weight of the robot. Example of applications are balancing  and stanTo Calculate Dynamic QuantitiesThe forces acting on a moving robot can be separated into two categories: forces exerted by contact and forces transmitted without contact . The CoP is linked to the former, and the ZMP to the latter. Nonetheless, it has been shown that both points coincide . TherefoThe CoP is defined as the point where the resultant force can be exerted with a zero resultant moment. When the contact is with a flat ground the CoP and ZMP, can be calculated as:Using the linear inverted pendulum model constraining the height of the CoM to be constant  or right (BNSR) depending on the support leg.Z-Torque: doing movements designed to generate torques around the z axis, while the robot is on a fixed pole.Based on availability and excitation of the sensor, two more types of datasets were studied.After adding this two dataset types the resulting 3D force space and 3D torque space with all types of datasets are depicted in Considering that most of the drift is assumed to be caused by temperature, it follows that by properly compensating the temperature drift, the offset of the sensor itself should be time invariant. This can be proven by using the offset estimated at the time of the calibration and applying it on some other time while the sensor is subjected to different temperatures.The result from the Model-based in situ calibration is a new calibration matrix. It is when using this calibration matrix that the improved measurements are obtained. Therefore, the new calibration matrix should be used somehow by the robot to obtain the improved measurements and better dynamic performance as a consequence.It is possible that is not easy to change the current calibration matrix of the sensor. In these cases, the proposed solution takes the form of a secondary calibration matrix. The secondary calibration matrix is the required transformation of the current calibration matrix to the new calibration matrix. It requires the knowledge of the current calibration matrix used by the sensor. It is calculated as follows:Before using the values obtained through force-torque sensing, they can be corrected by pre-multiplying with the secondary calibration matrix. This way the measurements used by the robot are the same as if the sensor was calibrated using the in situ calibration matrix.The contribution of the temperature can be added separately. In case no temperature calibration is available, these coefficients are loaded as zeros by default. This is helpful in cases where temperature measurements are available for some but not all sensors.Instead of estimating the offset before the experiment, the offset estimated by the calibration method can be used by summing the offset after the new calibration matrix and temperature compensation is applied.Workbench matrix are included as an estimation type in its own. At a first stage, the different dataset types were compared on their own. To further test the robustness of the in situ estimation, a different set of calibration and validation datasets were collected.The improvement in the measurements among the different estimation types is compared among the different kinds of possible calibration datasets to select the best way to improve the FT sensor performance. For comparison, results using the The validation datasets were taken on two different days, both different from the day the calibration datasets were collected. This was done to test the robustness to possible different ambient conditions. The datasets and their temperatures are showen in noTz, as indicated by name none of the Z-torque datasets were included.onlySupportLegs, from the balancing datasets, only the support leg was included. All other dataset types were included.AllGeneral, all dataset types were included.The calibration datasets were grouped into:The reasoning behind this arrangement of datasets is to see what combination of datasets provides the best results. Since it was proven before that Grid and Balancing together improve the calibration, the variables to test are the inclusion of Z-torque and non-support leg datasets.SnTdTO): Refers to the fact that the in situ offset removal is obtained by expecting a sphere in the force space when generating circular motions. No temperature considered.Sphere with no temperature (CnTdTO): Refers to the centralized offset removal method without considering temperature.Centralized with no temperature (OnTdTO): Refers to estimate the offset and the calibration matrix at the same time without considering temperature.One Shot with no temperature (SwTdTO): Refers to including temperature into the sphere type. But no temperature offset is considered.Sphere with temperature (CwTdTO): Refers to including the temperature into the centralized type. But no temperature offset is considered.Centralized with temperature (OwTdTO): Refers to estimate the offset and the calibration matrix at the same time considering temperature. But no temperature offset is considered.One Shot with no temperature (SwTrTO): Refers to including temperature into the sphere type. Removing temperature offset.Sphere with temperature (CwTrTO): Refers to including the temperature into the centralized type. Removing temperature offset.Centralized with temperature (OwTrTO): Refers to estimate the offset and the calibration matrix at the same time considering temperature. Removing temperature offset.One Shot with no temperature (Each strategy of offset estimation is considered an estimation type. Including temperature as a linear variable (wT) or not (nT) in the estimation are also considered different estimation types. If the temperature is considered, it is possible to take the first value as an offset (rTO) or not (dTO). Considering the three offset removal possibilities, adding the temperature as a linear variable to each of them and the temperature offset option results in the following nine estimation types:The logic behind the estimation type names can be seen in The evaluation could be roughly divided in three parts: one to observe the results of each estimation type, another to check the expected improvement on the robot of the generated calibration matrices and a third to verify the impact of using the contributions in a real robot. The sensor to calibrate is located near the hip of the left leg of an iCub robot.To understand better the behavior of the estimation types three comparisons are done. The first uses the mean square error (MSE) calculated between the force-torque data using the new calibration matrix and the model-based estimated data. A lower value would indicate a better fitting of the data. Mean Square Error (MSE) of each axis is calculated as follows:The selection of the best calibration matrix is done using the contact force validation described in a previous work . It emulC converge to Workbench matrix.The Contact force values when switching contact.Accepted gains values used in the low-level controller.Simultaneous online comparison of force torque measurements performance using different offsets.In most cases the dynamic behaviors of iCub are obtained through high-level controllers. They contain many tuning parameters that affect the behavior of the robot. The measurements of the sensor are used mainly in an indirect way by the high-level controllers. Therefore, finding quantitative measures of the improvement in the dynamic motions of the iCub caused by the in situ calibrated sensor is a challenging. Nonetheless, from the uses of the FT sensor on iCub, three quantitative methods were used:Contact force values when switching contactThe test performed consist in switching from single support to double support.Single support → double support → other single support.If the offset is calculated with the robot standing on one foot the sequence is:Double support → single support → double support → other single support.Instead if the offset is calculated with the robot in the air the sequence is:Since the robot is standing on flat ground it is expected that the only force acting on the robot feet is mass on the z axis. With no other force acting on the robot the forces in x and y should cancel each other in double support or be 0 when on single support. With this as a ground truth, is possible to evaluate the estimated contact forces at the feet when switching.Accepted gains values used in the low-level controllerThe robot was tuned to the maximum gain value in which the robot is able to perform the balancing demo in a satisfactory way. Beyond a certain value, the robot is observed to vibrate and fall. For this test, the high level gains of the controller are kept the same and only the low-level controller gains are changed. A higher gain value indicates that the robot is able to rely more on the measurements of the sensor as feedback. Thus, being able to use higher gains is better if it does not introduce unstable behaviors.Simultaneous online comparison of force torque measurements performance using different offsetsoffsetoffsetonline: is the offset calculated on the online on the robot the day of the experiment when the robot was just turned on. The temperature was 26 For this experiment the offsets used were estimated with a month difference with respect to the experiment date. The comparison between the expected FT value and the actual value obtained after applying both the offset and the secondary calibration matrix correction. Three different offsets where used:Three simultaneous estimation applications are launched. One for each type of offset used. The ground truth is calculated offline in the sections of the experiments in which there was only one contact with the environment. Multiple experiments were performed on the robot in the span of three hours to allow the FT sensors to heat up.To verify the behavior of the estimation types only the results from a single calibration dataset is showed. The one selected is the onlySupportLegs dataset. Nonetheless, the results extend to the other two calibration datasets.The MSE error of each estimation type is shown in Workbench matrix. In general, the highest values are obtained when comparing with the Workbench matrix. From these, the most different matrices are the ones obtained not considering temperature. Sphere type with no temperature (SnTdTO) is the most different of all with respect to the Workbench matrix.Is possible to see that the resulting calibration matrix from centralized types is the same as the one obtained through One shot types. The calibration matrix does not change between using the temperature offset (CwTrTO) or not (CwTdTO) for the centralized types.Workbench matrix as shown in Workbench matrix.Taking into account the Workbench matrix even for Workbench matrix, this similarity even when no penalization is used can be interpreted as a sign of better calibration. Considering this it can be seen that the sphere estimation types benefits from adding the temperature and even more from taking out the temperature offset. The centralized types benefit from adding the temperature, even if there seems to be no added benefit from considering the temperature offset.Is worth noticing that taking into account the temperature makes the matrix more similar to the The estimated offsets can be seen in The estimate offset varies more on the Centralized and Oneshot types. Is noteworthy that the fitting of the data is equal even if the offsets are different as seen from the MSE error in This validation was performed twice. One using only the calibration matrices estimating the offset in a few of the samples and the other using also the estimated offsets. The results of the contact force validation are shown in In this case, the offset is calculated taking some samples of the test experiments in which is known the robot is on one foot and not moving or moving slowly. The offset calculation includes not only the forces but also the temperature if coefficients are available.Workbench is considered to be correct when the sensor was unmounted, so becoming similar to it has benefits. On the other hand, the mounting changed the effectiveness of it, so being to close has lower performance. This is quite clear in Workbench for all the validation datasets.The best result is achieved by OwTdTO and OwTrTO with AllGeneral, When using the estimated offset, the best result is obtained by OwTrTO with Grouping the results by dataset, the calibration datasets can be ordered from best to worst in the following order: AllGeneral, onlySupportLegs and noTz. The behavior of a group of results by dataset is clear using the pallets of colors in These results are congruent with the space each dataset covers in the forces and torques 3D space. In Workbench matrix. From the difference in the results with respect to the Workbench is possible to see that the most affected axis by the mounting are By improving the measurements of the six-axis FT sensor through in situ calibration, it was possible to see improvements in the behavior of the floating base robot iCub.Results are shown in The balancing demo  was obseBased on the resulting coefficients of the temperature, Besides the quantitative evaluations of performance, an important qualitative behavior was observed when the measurements were used in high level controllers. During the experiments for the jerk control of floating base systems with contact-stable parametrised force feedback, it was observed that without the secondary calibration matrices the error in the estimated external forces was so big that the controller was unable to perform the experiments. Using the secondary calibration matrices reduced the error to ±2.5N ) in the Compared to other in situ calibration methods , the ModIn the previous Model Based formulation without temperature , less amIn the previous paper of model-based in situ calibrations with temperature compensation, results showed that the sphere types still had a certain advantage over centralized types. In those experiments, the amount of data used in calibration had less types of datasets equivalent to the noTz dataset here. By including the new types of datasets better results were obtained. Also, the one shot gave better performance than the sphere type. This shows a correlation between estimation type and types of calibration data used. The experiments described here show that datasets for calibration can be taken in different days in different conditions. This makes the data collection less restrictive despite the need for more data types. In the previous validation dataset, the offset was estimated at the beginning of the datasets. This is similar to the performance of the online estimated offset. This masked the difference in performance of the centralized offset with temperature and without. In here, the benefits of the method are further highlighted by showing the impact in the performance of floating base robots due to better FT measurements and the advantages of using the temperature are extended.The developed algorithm has been proven to improve the measurements of the sensor and the dynamic behavior of a floating base robot. It successfully accounts for temperature drift and can be extended to account for other lineal phenomena, allowing controllers to reliably use the sensor measurements as feedback, be it directly or indirectly.The new offset formulation is shown to give identical results as the centralized offset with the added benefit that the offset and calibration matrix are estimated simultaneously. The graphic representation of the sensor excitation in the 3D force and torque space has proven useful to provide intuitive insight into the comprehensive excitation of the sensor. In the The relationship between the regularization parameter and performance is clearly shown. This may allow to further refine and guide the selection of regularization parameters. The comparison with previous results suggests that the relevance and impact of each offset estimation strategy may be linked to the amount of data available. For small amounts of data, the physical assumption gives the highest improvements. With bigger amounts of data having no assumptions to estimate more accurately the calibration. The loss of effectiveness when using the physical assumption could be related to the fact that it does not consider temperature at all for offset estimation.As future work, ways to account for nonlinearities will be explored. Studying the dynamical response of the sensor is also interesting and might provide further improvements in the performance of the sensor.


Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3′ ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3′ ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3′ ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3′ end. Optimization of this activity is obtained through alteration of the lengths of the 5′-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3′ ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3′ end of T7 RNAP transcripts  There are two major forms of RNA editing, [A] substitutional, involving nucleotide modification, typically a transamination or deamination, which converts one nucleotide to another, for example, editing of tRNAs in Acanthamoeba castellanni2 or single nucleotide conversions such as A to I3), C to U or U to C4 and [B] insertional in which a nucleotide (or nucleotides) are inserted or deleted to produce a functional RNA7.RNA editing is an alteration of the genetic information in RNA relative to its DNA template. It is an additional step in gene expression, where specific nucleotides at specific positions are substituted for, added to, or deleted from the RNA in order to produce functional RNAs, especially mRNAs with a functional open reading frame. The term “RNA editing” was introduced in 1986 to describe the insertion of four uridine nucleotides into specific sites of mitochondrial cox2 mRNA in Trypanosome mitochondria12. Specificity of the nucleotide insertion or deletion site and the number of nucleotides inserted or deleted at a site is determined by an antisense RNA, termed a guide RNA [gRNA], which has the genetic information to produce functional mRNAs significantly different from the template DNA13.Two different types of insertional editing have been observed, post-transcriptional and co-transcriptional, each exemplified by a mitochondrial DNA system in which that type of insertional RNA editing is used extensively to produce functional mitochondrial RNAs. Post-transcriptional RNA editing, as exemplified by RNA editing in the mitochondria of trypanosomes, requires an endonuclease to break the RNA backbone at a specific site, a terminal transferase to add a nucleotide or nucleotides to the 3′ end of the RNA at the break site, and an RNA ligase to restore the RNA backbone at the site of the insertion or deletionPhysarum polycephalum produce functional mRNAs, rRNAs, and tRNAs17 different from their mtDNA template through non-templated addition of specific nucleotides at specific sites in nascent RNAs by the mitochondrial RNA polymerase (mtRNAP)20. Two major questions in the mechanism of this type of editing are: [A] How is the correct nucleotide inserted without a DNA template? [B] How is the correct site of insertion determined? Miller & Miller20 and Visomirski-Robic & Gott19 have each proposed that an unknown mechanism causes the mitochondrial RNA polymerase to pause during elongation of RNA on the DNA template, and insert a specific, non-templated nucleotide , before continuing DNA-templated elongation. Miller & Miller20 have shown that the Physarum mtRNAP is able to add nucleotides to the 3′ end of RNAs in the absence of DNA in vitro. However, with complex RNA populations without DNA templates in vitro any of the four ribonucleotides can be added.Co-transcriptional insertional RNA editing as exemplified by RNA editing in the mitochondria of myxomycetes such as 21 found in essentially all eukaryotic organisms and have conserved structural and functional similarities that indicate that they evolved from a common ancestor present at or near the advent of the eukaryotic line18. Interestingly, the conservation of these structural and functional similarities extend to other single subunit polymerases, such as the bacteriophage RNAPs24, DNA polymerases (DNAPs) such as the Klenow fragment of DNAP I in E. coli27, and reverse transcriptases (RNA-directed DNA polymerases) such as the HIV-1 reverse transcriptase30, constituting a super-family of single subunit polymerases with structural and functional homology.Both aspects of the specificity of this co-transcriptional RNA editing involve the mtRNAP. Mitochondrial RNAPs are single subunit polymerasesE. coli bacteriophage DNA and it has been extensively studied32. The crystal structure of T7 RNAP has been solved along with the different domains and their functions corresponding to both initiation and elongation processes35. It is a single subunit ~99 kDa protein (883 amino acid)36, found in Bacteriophage T7 and is primarily involved in transcription activity without the need of any other accessory transcription factors. The specificity and robustness of T7 RNA polymerase’s transcription activity comes from its specificity towards its promoter, a 23 nucleotide dsDNA sequence39. This specificity towards the promoter, involves the promoter recognition loop40 as well as the N terminus domain in T7 RNA polymerase42. T7 RNA polymerase can successfully carry out transcription on a synthetic ssDNA oligonucleotide template containing double stranded T7 promoter sequence44.The prototype RNA polymerase for this group of polymerases is the T7 RNAP encoded by the T7 de novo initiation which requires a promoter, single stranded DNA template, and the four ribonucleotide triphosphates; and elongation which requires a duplex with one strand having a recessed 3′ end (primer), the other strand 5′ extended (template), and four ribonucleotide triphosphates; there have been reports of a third activity in which addition of ribonucleotides occurs at the 3′ end of RNAs in the absence of a promoter or DNA template48. This activity was incorrectly attributed to the primer extension activity of elongation. Until recently, this activity of T7 RNAP has not been well characterized and most of the research on it has been focused on ways to prevent the addition of non-templated nucleotides to the 3′ ends of run-off RNAs50. Here we report the characterization of this activity. We show [A] that the T7 RNAP can add nucleotides to the 3′ end of RNAs and DNAs in a process that is neither elongation nor initiation, but is a type of RNA/DNA editing, [B] that the added nucleotide is added specifically as determined by base pairing of either an inter- or intramolecular template with an extended 5′ end relative to the recessed 3′ end, and [C] that the properties of the oligonucleotide that is provided determine the efficiency of nucleotide addition and distinguish this activity from either initiation or elongation activities. We will discuss this activity in terms of a general model of RNA editing by single subunit polymerases that may explain the specificity of nucleotide insertion during RNA editing by the Physarum mtRNAP.In addition to Synthetic single stranded DNA oligonucleotides  were synthesized by Eurofin-Operon. Oligonucleotides were dissolved in water to 200 µM and stored at 4 °C.Each oligonucleotide is named by a five number sequence separated by dashes where the first number is the oligonucleotide length, the second number is the length of the single stranded 5′ extension. The third and fifth number are the length of the double stranded region (stem of hairpin), and the fourth number is the length of the hairpin loop.T7 RNA polymerase (New England Biolabs (NEB) catalog number M0251S) was supplied in: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9), 1 mM EDTA, 20 mM 2-mercaptoethanol, 0.1% Triton X-100 and 50% glycerol and stored in a −20 °C freezer.2, 2 mM spermidine, 10 mM dithiothreitol, pH 7.9 @ 25 °C, 1 mg/ml BSA (Bovine Serum Albumin).NEB 10 × RNA pol reaction buffer was supplied. 1 × RNA Pol Reaction Buffer contains 40 mM Tris-HCl, 6 mM MgClin vitro oligonucleotide labeling protocol was derived from the in vitro transcription protocol recommended by New England Biolabs (NEB) for their T7 RNA polymerase51. For radiolabeling, (α-32P) rGTP, or (α-32P) rATP  were used. In vitro oligonucleotide radiolabeling was assayed in reactions containing 6 µl of 200 µM ssDNA oligonucleotides, 2 µl 10 × RNA polymerase buffer, 2 µl of 5 mM ribonucleoside triphosphates, and 1 µl of α-32P radiolabeled rATP/rGTP to a final volume of 20 µl. The reactions were incubated at 37 °C water bath for 60 minutes. After incubation 2 µl of 6× loading dye (10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 60% glycerol, 60 mM EDTA) was added to individual reaction mixtures and samples were run on 15% TBE (Tris-Borate EDTA) polyacrylamide gels at a constant 200 V and 75 mA, until the bromophenol blue migrated three-quarters of the length of the gel. Experiments were routinely repeated on both denaturing and non-denaturing gels to check their reproducibility.The TM. Detection of incorporated radioactivity was achieved by exposure to a phosphor screen (Molecular Dynamics) and scanned on a Phosphor Imager . Quantification of the phosphor image was done using ImageQuaNT software version 5.2 (Molecular Dynamics). Only the most intense band in each lane was quantitated. Graphical representation of major band intensities below each gel are shown to allow easier interpretation.Nucleic acids in the gels were stained with 1× TBE, Ethidium Bromide for 10 min and then visualized under Bio-Rad Gel Doc52 and that T7 RNAP could add ribonucleotides to the 3′ end of DNA and RNA55. We hypothesized that these observations indicated that these RNAPs had a novel activity similar to the RNA editing observed in mitochondria of Physarum polycephalum7 and other myxomycetes56. To study this activity, we have developed an in vitro assay for labeling the 3′ ends of RNA and DNA oligonucleotides using T7 RNAP and a single radiolabeled ribonucleotide triphosphate. In contrast to using complex mixtures of RNA or DNA, with these simple oligonucleotide sequences, some oligonucleotides failed to label while others labeled efficiently. We have determined that this labeling specificity depends on the ability of the 3′ end of the oligonucleotide to anneal with an internal complementary sequence which positions the 3′ end next to a nucleotide that is not base paired in the 5′ extension of the oligonucleotide. This can take place within the same oligonucleotide (intramolecular base pairing) or between complementary sequences in two separate oligonucleotides (intermolecular base pairing) as shown in Fig. Several laboratories have reported that single subunit RNAPs could add radiolabeled nucleotide triphosphates to the 3′ end of RNA in the absence of a DNA templateAn example of these experiments is shown in Fig. This experiment demonstrates that when presented with an oligonucleotide with a 3′ end with the potential to base pair as a recessed 3′ end, either inter- or intra- molecularly, the T7 RNAP is able to add the radiolabeled nucleotide to the 3′ end, but only if it can form a Watson-Crick base pair with the nucleotide in the 5′ extension (template) next to the nucleotide base paired with the 3′ end nucleotide (primer).To better understand this self-templated labeling at the 3′ end and to optimize this reaction, the single stranded DNA oligonucleotides have been systematically altered to determine the importance and contribution of [1] the overall length, [2] the length and base composition of the potential duplex, [3] the length of the 5′ extension, and [4] loop length (number of nucleotides separating the complementary sequences) with the goal of optimizing the reaction.To determine the contribution of 5′ extension length, oligonucleotides were designed with increasing 5′ extension lengths (0–16 nucleotides), constant base pairing length (6 pairs), constant loop length (8 nucleotides), and increasing overall length (20–36 nucleotides).Figure To determine the contribution of loop length to labeling efficiency, oligonucleotides were designed with constant 5′ extensions (2 nucleotides), constant double stranded regions (either 9 nucleotides or 6 nucleotides), and variable loop lengths (6–24 nucleotides) and variable overall length (20 nucleotides to 44 nucleotides). Nucleotide labeling efficiencies are shown in Fig. Labeling is seen for oligonucleotides with both intramolecular and intermolecular base pairing. Figure To determine the contribution of the length of the base pairing to 3′ end labeling efficiency, the base pairing length was systematically altered while keeping the overall length (32 nucleotides) and the 5′ extension length (2 nucleotides) constant  and the 5′ extension constant (at either 2 nucleotides or 10 nucleotides) constant. The V-10-V-8-V series is shown in Fig. To determine if this optimum base pairing length for labeling efficiency also pertained to intermolecular base pairing and to eliminate the contribution of loop length to labeling efficiency, a series of oligonucleotides with varying lengths of sequence complementary to a template oligonucleotide were designed. Figure Below the template strands in Fig. Figure Figure On the right side of Fig. While overall oligonucleotide length does not correlate with labeling efficiency, experiments to determine the optimum length of the 5′ extension, the double stranded region (stem), and the loop for the efficiency of labeling the 3′ end with a specific nucleotide give optimum lengths for one of the parameters when the other two parameters are held constant. However, the optimums for a parameter do not appear be independent, since a different optimum for a parameter is observed when the other two parameters are fixed at different lengths. To determine the interdependence of the three parameter lengths, 27 oligonucleotides were created with 3, 6, or 9 nucleotides in the 5′ extension, double stranded region, or loop in all possible combinations Fig. . The relAnalysis of these results allows several generalizations to be made about the interdependence of the parameter lengths. Labeling efficiency generally increases with 5′ extension length for a given stem length/loop length. However, different stem length/loop length combinations give significantly different efficiencies with 3, 6, or 9 nucleotides in the 5′ extension.The labeling efficiencies generally either decrease with the length of the double stranded stem region (3 base pairs >6 base pairs >9 base pairs) for six 5′ extension/loop combinations or peak with 6 base pairs (6 base pairs >3 or 9 base pairs) for the three 5′ extension/loop combinations with loops of 9 nucleotides.Labeling efficiencies vary with loop length in several different ways. For the three oligonucleotides with 6 base pair stems, 9 nucleotides loops have the highest labeling efficiency (9 nucleotides >3 or 6 nucleotides). For the three oligonucleotides with 3 base pair stems, 9 nucleotide loops have the lowest efficiency (3 or 6 nucleotides >9 nucleotides). For the remaining three oligonucleotides with 9 base pair stems, 6 nucleotide loops have the lowest labeling efficiency (3 and 9 nucleotides >6 nucleotides or 3 nucleotides >6 or 9 nucleotides).The highest labeling efficiencies are with oligonucleotides 12-3-3-3-3 and 27-6-6-9-6 followed closely by 30-9-6-9-6, 15-6-3-3-3, and 18-9-3-3-3. Oligonucleotides in the X-X-3-3-3 series have high labeling efficiency, essentially independent of 5′ extension length. Oligonucleotides of the X-X-6-9-6 series have significantly higher labeling efficiencies with 6 or 9 nucleotide (longer) 5′ extensions.Since the formation of a base paired region (stem) is necessary to put a recessed 3′ primer end on the 5′ extension template, it is surprising that longer stem lengths generally give decreased labeling efficiencies. While at least three nucleotides of base pairing complementarity are needed for significant labeling, labeling efficiencies with increasingly longer base pairing potential generally increase up to a peak of about 5 – 7 base pairs and then decline with increasing length, where loop length and 5′ extension length are held constant. This duplex optimum could be caused by at least two possible constraints, [A] a duplex length constraint (static model) in which only duplexes of intermediate length can be bound productively, and [B] a duplex stability constraint (dynamic model) where a duplex is required but where labeling efficiency decreases with duplex stability, indicating that both single stranded and duplex forms are necessary.Efficiency probably does not depend directly on duplex length since different loop lengths and 5′ extension lengths, when held constant, give a different optimum duplex length , constant 5′ extension (10 nucleotides), and duplex regions varying from 3 nucleotides to 7 nucleotides and from 7 to 18 hydrogen bonds Figs  and S1. 3G2T, A3GCT, A2G2T2) and two similar base composition with 15 hydrogen bonds (AG3T and A2G3T) were compared. The four oligonucleotides with base composition A3G2T or A3GCT varied in relative labeling efficiency from 86% to 100%, and the four oligonucleotides with base composition A2G2T2 varied in relative labeling efficiency from 9% to 100%. Likewise, the four oligonucleotides with base composition AG3T varied in relative labeling efficiency from 48–95%. These wide variations in relative labeling efficiency with oligonucleotides with the same base composition in their duplex eliminated base composition as a factor in relative labeling efficiency.In order to determine the cause of the variation in relative labeling efficiency within oligonucleotides with duplexes with the same number of hydrogen bonds, we compared relative labeling efficiencies of oligonucleotides with different base compositions but with the same number of hydrogen bonds in the duplex. There are 15 different base compositions of 6 nucleotides with 14 hydrogen bonds, and there are 16 different base compositions of 6 nucleotides with 15 hydrogen bonds. Three similar six base compositions with 14 hydrogen bonds  than oligonucleotides in which the G nucleotides in the duplex were separated by other nucleotides (9% to 48%).This paper describes and characterizes a novel DNA/RNA editing activity of T7 RNAP with nucleotide specificity. This novel activity is detected when an oligonucleotide is provided that has a duplex with a recessed 3′ end (primer) and a 5′ extension (template) where the first unpaired base specifies the labeling nucleotide through Watson-Crick base pairing. This novel editing activity differs from the two classic transcription activities, initiation and elongation. It differs from initiation in that nucleotide addition occurs in the absence of a promoter, and differs from elongation in that nucleotides are not added processively on the template, but only one or a few nucleotides are added since each nucleotide added to the duplex ultimately decreases the labeling efficiency. Probably the most unique feature of this novel activity is the addition of a DNA templated ribonucleotide to a DNA 3′ end to form a DNA primed, RNA-DNA duplex. Although T7 RNAP will only add ribonucleotides to 3′ ends to create RNA strands, it is able to add ribonucleotides to 3′ ends of either RNA or DNA and will use either RNA or DNA templates to specify ribonucleotide addition. Therefore, either RNA or DNA oligonucleotides able to form intramolecular and intermolecular duplexes with recessed 3′ ends can be specifically labeled with radiolabeled ribonucleotides as well as DNA oligonucleotide templates with RNA primers with recessed 3′ ends or RNA oligonucleotide templates with DNA primers with recessed 3′ ends.57 and other single subunit RNAPs55. These reports did not mention the potential for nucleotide specificity presumably because they were using complex templates that positioned 3′ recessed ends at many positions, and therefore, next to essentially any nucleotide. Rather than studying this activity, most of the focus was on blocking non-templated nucleotide addition which produced in vitro transcripts with unintended sequences. A few studies using less complex templates demonstrated that T7 RNAP could add nucleotides to RNA in the absence of DNA, presumably through inter- and intramolecular formation of RNA-RNA duplexes48. Konarska and Sharp44 have reported ribonucleotide addition to RNA templated, RNA primed oligonucleotides but did not explore the mechanisms of this activity and did not report activity on DNA templated or DNA primed oligonucleotides. Krupp45 observed promoter independent RNA synthesis on DNA templates able to form intermolecular or intramolecular hairpin loops that could be used as DNA primers for RNA synthesis forming RNA-DNA duplexes. However, none of these reports discussed the actual mechanism and criteria for T7 RNAP to add these apparently non DNA-templated nucleotides to the 3′ end. These reactions were all done with four ribonucleotide triphosphates present and often produced complex gel patterns that were complicated to interpret. Here we used DNA oligonucleotides with a single radiolabeled ribonucleotide triphosphate present to simplify interpretation.There have been a few reports of non-templated nucleotide addition to 3′ ends of nucleic acids with T7 RNAPTo determine their contribution to labeling efficiency, we have systematically varied the three parts of the oligonucleotide . In general, labeling efficiency increases with 5′ extension length up to about 10 nucleotides. The loop length can affect labeling efficiency depending on duplex length, but is essentially optional since intermolecular base pairing between two separate oligonucleotides can also produce 3′ end labeling. Small loops when present seem to destabilize a duplex and longer than optimal duplexes can be activated by a small loop while optimal length duplexes may be inactivated by a short loop. On the other hand, longer loops may be detrimental since they increase the distance between the component strands of the duplex decreasing the probability of duplex formation. The most critical portion of these oligonucleotides is the duplex which positions the 3′ end recessed relative to the 5′ end. The duplex length for optimal labeling is about six base pairs for duplexes 33% GC rich (14 hydrogen bonds), about 9 nucleotides at 0% GC (18 hydrogen bonds), or about three base pairs at 66% GC (8 hydrogen bonds).The observation that duplex stability is a major factor in labeling efficiency implies a dynamic, two-step mechanism for labeling. Although the duplex must transiently form in order to prime for labeling, duplex instability implies that an unfolded single strand intermediate is required for polymerase binding. If the polymerase binds the unfolded single strand oligonucleotide in a productive way, so that the duplex can form with the 3′ end base paired and at the active site of the T7 RNAP, a nucleotide complementary to the template base binds, and a phosphodiester bond is formed incorporating the base into the duplex. In this model, phosphodiester bond formation is coupled to the release of the T7 RNAP. This non-processive mechanism is in contrast to elongation which uses a processive, primer extension on template mechanism. Although duplex stability appears to be a major factor in labeling efficiency, additional sequence motifs may enhance RNA polymerase binding and therefore contribute to labeling efficiency. One motif that seems to enhance 3′ end labeling in oligonucleotides is consecutive G nucleotides (GG motif).de novo by binding to a promoter and then transitioning to elongation through promoter clearance. Early RNAPs probably lacked promoter recognition domains and initiated through primer extension like their closely-related DNA polymerases or through some other non-promoter mechanism. Several contemporary examples of such RNA polymerases exist52. It is likely that the RNA/DNA editing activity of T7 RNAP derives from such a primal activity. Mutations in the gene for T7 RNAP produce altered enzymes which support this idea. For example, some mutated enzymes have been found to be catalytically active although they bind promoter weakly, and in some instances mutated enzymes which are transcriptionally inactive have strong binding to the promoter58. Also, mutated T7 RNA polymerase59 with two amino acid substitutions (Y639F and S641A) has altered specificity towards promoter, but gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA60. This Y639F mutant retains the ability to use RNA or DNA templates and can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction55.One of the major differences between RNA polymerases and DNA polymerases is the ability of RNAP to initiate synthesis 61. One of the major questions about Myxomycete RNA editing is how the identity of the inserted nucleotide is determined. Based on the observed in vitro mechanism of editing in T7 RNAP, we propose a model of nucleotide specificity in RNA editing for the related Physarum mtRNAP. At an RNA editing site, the Physarum mtRNAP pauses and then may either stay in place, release, or back up on the template to allow the 3′ end of the RNA to loop back on a complementary sequence either in the RNA or in the non-template strand of DNA, and add a nucleotide at the 3′ end. This positioning of the 3′ end allows specific addition to the 3′ end using the vacant position next to the 3′ end to specify the added nucleotide through Watson-Crick base pairing. The transient base pairing of the RNA is then released resulting in the insertion of a nucleotide at the 3′ end not templated by the template strand of DNA. Elongation is then resumed as a phosphodiester bond is formed between the unpaired 3′ nucleotide and the next templated nucleotide. In vitro presentation of the Physarum mitochondrial RNAP (mtRNAP) with oligonucleotides, similar to those described above, would be a test of this hypothesis. Confirmation of this novel activity in Physarum mtRNAP would be a major step toward understanding the co-transcriptional mechanism of RNA editing by the mtRNAP in mitochondria of myxomycetes.This novel DNA/RNA editing activity in T7 RNAP is probably present in all single subunit polymerases and may be the basis of nucleotide specificity during RNA editing by the mtRNAP in mitochondria of myxomycetesSupplementary Information


Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major  Telomeres are DNA-protein complexes that protect the ends of linear eukaryotic chromosomes. In most species telomeric repeats are synthesized by telomerase consisting of reverse transcriptase and an RNA template. Telomere-associated proteins form the telomere protection shelterin complex . RecentlDrosophila we conducted a systematic study of the biogenesis and function of telomeric transcripts and their associated proteins in the female germline and in early development. Telomerase was not found in the Drosophila genome [Drosophila telomeres is that they are composed of LINE (long interspersed nuclear element) retrotransposons; namely, HeT-A, TART and TAHRE, among which HeT-A is the most abundant [Drosophila and species encoding telomerase, the basic mechanisms of telomere maintenance are similar [Drosophila telomeric protein complex is structurally different from human shelterin, but is functionally analogous and protects the chromosome ends from degradation and fusion [Drosophila were described a decade earlier than human TERRA [Drosophila telomeric transcriptome in the germline consists of both long retrotransposon transcripts and small RNAs [HeT-A and TART produce multiple sense and antisense transcripts, the latter containing multiple introns [HeT-A and TART retrotransposons drive the bidirectional transcription [HeT-A sense transcripts may be considered as functional analogues of both the telomerase RNA template and TERRA RNA. Clearly, the expression of telomeric elements must be strictly controlled to ensure regulation of telomere length. In Drosophila ovaries, such control is mediated by a distinct mechanism of RNA interference, the piRNA (PIWI interacting RNA) pathway. In the germline, antisense transcripts of telomeric retrotransposons serve as piRNA precursors, regulating the abundance of sense coding transcripts [HeT-A expression in the germline leading to severe developmental defects [Using a genome . A uniquabundant . Despite similar . Drosophd fusion , 10. Traan TERRA , 12. Theall RNAs , 14. HeT introns . Unusualcription , 17. HeTnscripts , 14. In nscripts . piRNA p defects –21.Drosophila telomere biology is whether the level of HeT-A RNA could play a role in cellular response to the state of telomeres. To address this question, it is critical to examine the HeT-A RNA and HeT-A Gag biogenesis in normal development and with telomere dysfunctions.Telomeres have certain features of heterochromatin, therefore, their transcriptional activity in a normal cell is repressed. Increased level of telomere transcription in response to telomere damage may be a part of telomere signaling affecting various cellular processes . An impoHeT-A expression, abundant long telomeric RNAs accumulate in the germline, then transported to the oocyte and form aggregates at the mitotic spindles during the syncytial stage of embryogenesis [HeT-A RNA and Gag protein in the germline and early development upon piRNA pathway disruption.Previously, we have shown that the mechanisms of chromosome end protection are closely related to the silencing of telomeric repeats: upon depletion of the piRNA pathway components and other factors suppressing ogenesis . This phogenesis , 21. HowHeT-A and TART were detected at different stages of germline development in wild type Drosophila strains as well as upon depletion of telomere silencing components [Drosophila telomeric RNP in the germline and in early development is still poorly understood since most studies are focused on either HeT-A and TART RNA or their protein products separately.RNA-binding proteins are involved in RNA metabolism at different stages of its life cycle, from transcription to degradation. Transcripts and proteins encoded by mponents , 23, 24.Gaiano strain which is characterized by increased HeT-A and TART copy numbers [HeT-A Gag protein is barely detected in germ cells [HeT-A piRNAs in Gaiano ovaries [Drosophila telomeric RNP were recently obtained from studies of somatic tissues. HeT-A spherical particles consisting of HeT-A Gag and HeT-A RNA were discovered in proliferating neuroblasts where they associate with telomeres undergoing DNA replication and are considered a prerequisite for somatic HeT-A transposition [Due to robust piRNA-mediated silencing in the germline, telomeric retrotransposon products are present at very low levels in wild type ovaries. Even in the  numbers , HeT-A Grm cells , which c ovaries . Valuablposition .HeT-A transcripts interact with the RNA-binding protein Gag-HeT-A, which they encode, and that they form RNP particles during most of their life cycle in oogenesis and early embryogenesis. In germarium, HeT-A RNP form spherical structures in the nuclei of dividing cystoblasts and seem to be the intermediates of telomere elongation in the germline. It has been shown that interaction of HeT-A RNPs with the main carrier protein Egalitarian (Egl) [HeT-A RNP in early development. Retention of Egl at HeT-A RNP causes its ectopic localization at syncytial stage of embryogenesis which likely impairs embryonic development in the piRNA pathway mutants.Here, we show that upon piRNA loss overexpressed an (Egl)  is requiHeT-A element encoding Gag protein tagged with HA (hemagglutinin) and FLAG epitopes was cloned into pUASp-attB as described previously [HeT-A 3`UTR. The construct pUAST-attB-HeTA-HA-FLAG-mut_ms2 was created on the basis of pUAST-attB-HeTA-HA-FLAG-ms2 by cloning of the mutated HeT-A hairpin (Germline Knockdown) flies were F1 progeny of the genetic cross of a strain bearing a construct with short hairpin (sh) RNA  and a driver strain #25751 . The transgenic strain expressing fused protein Egl-GFP was kindly provided by S. Bullock [Gaiano III (GIII) is a strain carrying a third chromosome with Tel locus derived from natural Gaiano strain characterized by extremely long telomeres [Full-length eviously . To gene hairpin . Constru Bullock . The Gai2, 0.5% NP-40, Complete mini protease inhibitor cocktail (Roche), 20 mM NaF, 0.2 mM NaVO4, 1 U/μl RiboLock RNase Inhibitor (ThermoScientific)). For RIP with anti-HA beads, lysis buffer was supplemented with 1% Triton X-100, 0.1% SDS and 10% glycerol. The extracts were cleared by centrifugation at 16,000 g at 4° C for 10 min and diluted with 9 volumes of NT2 buffer . The lysates were incubated with anti-HA magnetic beads (Pierce) or with antibody-coated (5 μg of the primary antibodies per sample) Dynabeads Protein A (Invitrogen) for 2 h at 4°C on a rotator. After 3 washes in NT2 buffer, 1/10 of beads were saved for Western blot analysis. RNA was isolated from the remaining beads using Trizol reagent (Life Technologies). Reverse transcription was performed with a random hexanucleotide primer and SuperScriptIII reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. qPCR was performed on a LightCycler96 (Roche). Primers are listed in RIP was performed according to  with modHeT-A riboprobe containing a fragment of the ORF (nucleotides 4330–4690 of GenBank sequence DMU06920) was used. The specificity of this probe was previously confirmed by Northern blotting and in situ hybridization with polytene chromosomes of salivary glands [RNA FISH combined with immunostaining was carried out according to the previously described procedure  with mody glands , 26. To 2, 0.1% Triton X-100, Complete Mini protease inhibitor cocktail (Roche), 0,2 mM NaVO4, 20 mM NaF, RiboLock 1U/μl (ThermoScientific)). The extracts were cleared by centrifugation at 16,000 g at 4°C for 10 min. Supernatants were incubated with anti-HA magnetic beads  for 30 min at room temperature on a rotator. After 3 washes in IP buffer the bound proteins were eluted from the beads by boiling in 100 μl of Laemmli protein loading buffer  for 5 min. Beads were pelleted and the supernatant was saved for Western blot analysis. Samples were resolved on 8% SDS-PAGE gel and transferred onto Immobilon-P PVDF membrane (Millipore). Blots were developed using the Immun-Star AP detection system (Bio-Rad Laboratories), in accordance with the manufacturer’s recommendations.To prepare embryonic extracts, 0-2-hour old embryos were dechorionated, frozen and stored at -80°C. Ovaries (400 pairs) or embryos (the volume of dechorionized embryos ~70 μl) were homogenized in a Dounce homogenizer in 9 volumes of cold IP Buffer  and is referred to as cis-preference [Drosophila telomeric protein Gag, encoded by HeT-A, interacts with HeT-A RNA forming RNP in the germline. Recombinant engineering of epitope tags and inducible tissue-specific expression of transgene containing full-length HeT-A have made detection and purification of HeT-A Gag complexes routine [HeT-A is strongly repressed in the germline [HeT-A Gag is not detectable by Western blotting in the ovaries of GIII and transgenic strains expressing HeT-A-HA in contrast to somatic tissues  combined with immunostaining. In the germarium region 2A, HeT-A transcripts co-localize with Gag-positive particles forming spherical RNPs of 1–2 microns in size  we took advantage of another transgenic strain containing a HeT-A copy, tagged by the MS2 bacteriophage hairpins, and encoding for Gag-HA. Combined MS2 RNA FISH and HA-immunostaining shows colocalization of transgenic RNA and protein in the nuclei and cytoplasm of germ cells , oskar (osk) and gurken (grk) mRNAs are associated with Egl-BicD complex in wild type ovaries  is presumably involved in the HeT-A RNA localization, and therefore, generated а transgenic strain, encoding HeT-A transcripts with impaired hairpin structure  by Western blotting. Extracts were prepared from the GIII strain (first lane) and transgenic strains expressing UAS-HeT-A-HA in the germline on a wild type background (second lane) or upon spnE_GLKD (third lane). Antibodies are indicated to the left. In ovaries, HeT-A Gag is detected only upon piRNA pathway disruption (spnE_GLKD).  HeT-A RNA FISH (green) combined with endogenous HeT-A Gag (red) immunostaining on ovaries of GIII strain. HeT-A RNPs are not reveled in the germ cells but detected in somatic cells of germarium  in accordance to previously published observation (23). Intensive HeT-A staining observed in the nurse cell nuclei appear to be correspond to the transcribed telomeres (arrows) (C).(A) Detection of (TIF)Click here for additional data file.S2 Fig(A)HeT-A RNA (green) and HeT-A Gag-HA (red) form HeT-A RNPs (arrows) in the cytoplasm of nurse cells in the ovaries of nosGal4; UAS-HeT-A-HA; UAS-spnE_sh flies. Egg chamber at stage 7 of oogenesis is shown. (B) Colocalization of HeT-A RNA (green), HeT-A Gag-HA (red) and telomeric protein HipHop (magenta) is indicated by arrows. Nurse cell nucleus of a stage 6 is shown. (C) HeT-A RNA FISH (green) combined with endogenous HeT-A Gag (red) immunostaining on ovaries of non-transgenic spnE_GLKD strain. A fragment of a stage 7 egg chamber is shown. (D) HeT-A RNA FISH (green) combined with HeT-A Gag (red) immunostaining was performed on ovaries of yw wild type strain. An egg chamber at stage 7 of oogenesis is shown. DNA is stained with DAPI (blue).(TIF)Click here for additional data file.S3 FigHeT-A Gag-HA (green) (A) or Egl (green) (B) is shown. Stage 6 egg chambers of transgenic strains expressing UAS-HeT-A-Gag-HA in the germline in wild type background (bottom panels) or upon spnE_GLKD (top panels). Arrows indicate nuage. Egl colocalizes with Vasa in nuage, while HeT-A Gag-HA staining is more diffuse and only partially overlapped with Vasa. Magnification is 63x.Immunostaining of a nuage component Vasa (red) and (TIF)Click here for additional data file.S4 Figrp49 was used for normalization. Western blot analysis of co-immunoprecipitated proteins is shown to the right. The antibodies used for Western blotting are indicated to the right. The antibodies used for co-IP are indicated above the IP lanes. Lane designation: input , pellet (insoluble fraction), IP (precipitates). Anti-Egl immunoprecipitates both Egl and BicD proteins but not HeT-A Gag which is enriched in insoluble fraction. (B) RT-qPCR analysis of RNA precipitated with anti-GFP relative to negative control  from ovary lysates of w; tub-Egl-GFP flies. Western blot analysis of co-immunoprecipitated proteins is shown to the right; the indications are as in (A). Anti-GFP immunoprecipitates both Egl-GFP and Egl as well as BicD indicating that Egl-BicD is an oligomeric complex. For RIP panels, the error bars represent SEM of 2 biological replicas.(A) RT-qPCR analysis of RNA precipitated with anti-Egl relative to negative control  from ovary lysates of nosGal4; UAS-HeT-A-HA; UAS-spnE_sh flies. (TIF)Click here for additional data file.S5 FigHeT-A RNA (green), HeT-A Gag-HA (magenta) and Egl (red) form granules (arrows) in the cytoplasm of nurse cells in ovaries of nosGal4; UAS-HeT-A-HA; UAS-spnE_sh flies. Egg chamber at stage 7 of oogenesis is shown. (B) Egl (red) and endogenous HeT-A Gag (green) form granules (arrows) in ovaries of spnE_GLKD not carrying UAS-HeT-A-HA transgene. DNA is stained with DAPI (blue). (C) Co-IP of HeT-A Gag. Western blot analysis of proteins immunoprecipitated with anti-Gag HeT-A from ovaries of spnE_GLKD flies. Anti-Gag HeT-A immunoprecipitates Egl protein. The antibodies used for Western blotting are indicated to the right and the antibodies used for co-IP are indicated above the IP lanes.(A) (TIF)Click here for additional data file.S6 FigHeT-A localization signal 1) is revealed in the 3’UTR of HeT-A copies . Folding of HLS1 is shown. (B) Sequence of mutated HeT-A hairpin is shown. (C) Colocalization of MS2 RNA (green), HeT-A Gag-HA (magenta) and Egl (red) in the cytoplasm of nurse cells (arrowheads) and in the oocyte (arrows) in ovaries of nosGal4; UAS-HeT-A-HA-ms2_mut flies upon spnE_GLKD. Two egg chambers at different stages of oogenesis are shown. (D) MS2 RNA FISH (green) combined with immunostaining of gamma-tubulin (red) was performed on 0-2-hour old embryos of nosGal4; UAS-HeT-A-HA-ms2_mut flies upon spnE_GLKD. DNA is stained with DAPI (blue). Syncytial metaphase is shown. (E) HeT-A Gag-HA (red) and Egl (green) immunostaining were performed on 0-2-hour old embryos of nosGal4; UAS-HeT-A-HA-ms2_mut flies upon spnE_GLKD. DNA is stained with DAPI (blue).(A) A putative HLS1 ((TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.