LINE-1 mRNA 3' end dynamics shape its biology and retrotransposition potential.

LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'→3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.

Kinesin KIF18A is a novel PUM-regulated target promoting mitotic progression and survival of a human male germ cell line.

Regulation of proliferation, apoptosis and cell cycle is crucial for the physiology of germ cells. Their malfunction contributes to infertility and germ cell tumours. The kinesin KIF18A is an important regulator of those processes in animal germ cells. Post-transcriptional regulation of KIF18A has not been extensively explored. Owing to the presence of PUM-binding elements (PBEs), KIF18A mRNA is a potential target of PUM proteins, where PUM refers to Pumilio proteins, RNA-binding proteins that act in post-transcriptional gene regulation. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assays, by applying an appropriate luciferase construct encoding wild-type KIF18A 3'-UTR, upon PUM overexpression or knockdown in TCam-2 cells, representing human male germ cells. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function, an MTS proliferation assay, and apoptosis and cell cycle analysis using flow cytometry, was performed upon KIF18A mRNA siRNA knockdown. KIF18A significantly influences proliferation, apoptosis and the cell cycle, with its effects being opposite to PUM effects. Repression by PUM proteins might represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells.

Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model.

Nanos RNA-binding proteins are critical factors of germline development throughout the animal kingdom and their dysfunction causes infertility. During evolution, mammalian Nanos paralogues adopted divergent roles in germ cell biology. However, the molecular basis behind this divergence, such as their target mRNAs, remains poorly understood. Our RNA-sequencing analysis in a human primordial germ cell model-TCam-2 cell line revealed distinct pools of genes involved in the cell cycle process downregulated upon NANOS1 and NANOS3 overexpression. We show that NANOS1 and NANOS3 proteins influence different stages of the cell cycle. Namely, NANOS1 is involved in the G1/S and NANOS3 in the G2/M phase transition. Many of their cell cycle targets are known infertility and cancer-germ cell genes. Moreover, NANOS3 in complex with RNA-binding protein PUM1 causes 3'UTR-mediated repression of FOXM1 mRNA encoding a transcription factor crucial for G2/M phase transition. Interestingly, while NANOS3 and PUM1 act as post-transcriptional repressors of FOXM1, FOXM1 potentially acts as a transcriptional activator of NANOS3, PUM1, and itself. Finally, by utilizing publicly available RNA-sequencing datasets, we show that the balance between FOXM1-NANOS3 and FOXM1-PUM1 expression levels is disrupted in testis cancer, suggesting a potential role in this disease.

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5'-Terminal Region of p53 mRNA.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5' terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3' end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5' terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

Regulation of the p53 expression profile by hnRNP K under stress conditions.

The p53 protein is one of the transcription factors responsible for cell cycle regulation and prevention of cancer development. Its expression is regulated at the transcriptional, translational and post-translational levels. Recent years of research have shown that the 5' terminus of p53 mRNA plays an important role in this regulation. This region seems to be a docking platform for proteins involved in p53 expression, particularly under stress conditions. Here, we applied RNA-centric affinity chromatography to search for proteins that bind to the 5' terminus of p53 mRNA and thus may be able to regulate the p53 expression profile. We found heterogeneous nuclear ribonucleoprotein K, hnRNP K, to be one of the top candidates. Binding of hnRNP K to the 5'-terminal region of p53 mRNA was confirmed in vitro. We demonstrated that changes in the hnRNP K level in the cell strongly affected the p53 expression profile under various stress conditions. Downregulation or overexpression of hnRNP K caused a decrease or an increase in the p53 mRNA amount, respectively, pointing to the transcriptional mode of expression regulation. However, when hnRNP K was overexpressed under endoplasmic reticulum stress and the p53 amount has elevated no changes in the p53 mRNA level were detected suggesting translational regulation of p53 expression. Our findings have shown that hnRNP K is not only a mutual partner of p53 in the transcriptional activation of target genes under stress conditions but it also acts as a regulator of p53 expression at the transcriptional and potentially translational levels.

PUM1 and PUM2 exhibit different modes of regulation for SIAH1 that involve cooperativity with NANOS paralogues.

Pumilio (PUM) proteins are RNA-binding proteins that posttranscriptionally regulate gene expression in many organisms. Their PUF domain recognizes specific PUM-binding elements (PBE) in the 3' untranslated region of target mRNAs while engaging protein cofactors such as NANOS that repress the expression of target mRNAs through the recruitment of effector complexes. Although the general process whereby PUM recognizes individual mRNAs has been studied extensively, the particulars of the mechanism underlying PUM-NANOS cooperation in mRNA regulation and the functional overlap among PUM and NANOS paralogues in mammals have not been elucidated. Here, using the novel PUM1 and PUM2 mRNA target SIAH1 as a model, we show mechanistic differences between PUM1 and PUM2 and between NANOS1, 2, and 3 paralogues in the regulation of SIAH1. Specifically, unlike PUM2, PUM1 exhibited PBE-independent repression of SIAH1 3'UTR-dependent luciferase expression. Concordantly, the PUF domains of PUM1 and PUM2 showed different EMSA complex formation patterns with SIAH1 3'UTRs. Importantly, we show direct binding of NANOS3, but not NANOS2, to SIAH1 3'UTR, which did not require PBEs or the PUF domain. To the best of our knowledge, this is the first report, showing that an NANOS protein directly binds RNA. Finally, using NANOS1 and NANOS3 constructs carrying mutations identified in infertile patients, we show that these mutations disrupt repression of the SIAH1-luciferase reporter and that the central region in NANOS1 appears to contribute to the regulation of SIAH1. Our findings highlight the mechanistic versatility of the PUM/NANOS machinery in mammalian posttranscriptional regulation.

High-density surface electromyography maps after computer-aided training in individual with congenital transverse deficiency: a case study.

BACKGROUND: The aim of this study was to determine whether computer-aided training (CAT) of motor tasks would increase muscle activity and change its spatial distribution in a patient with a bilateral upper-limb congenital transverse deficiency. We believe that our study makes a significant contribution to the literature because it demonstrates the usefulness of CAT in promoting the neuromuscular adaptation in people with congenital limb deficiencies and altered body image. CASE PRESENTATION: The patient with bilateral upper-limb congenital transverse deficiency and the healthy control subject performed 12 weeks of the CAT. The subject's task was to imagine reaching and grasping a book with the hand. Subjects were provided a visual animation of that movement and sensory feedback to facilitate the mental engagement to accomplish the task. High-density electromyography (HD-EMG; 64-electrode) were collected from the trapezius muscle during a shrug isometric contraction before and after 4, 8, 12 weeks of the training. After training, we observed in our patient changes in the spatial distribution of the activation, and the increased average intensity of the EMG maps and maximal force. CONCLUSIONS: These results, although from only one patient, suggest that mental training supported by computer-generated visual and sensory stimuli leads to beneficial changes in muscle strength and activity. The increased muscle activation and changed spatial distribution of the EMG activity after mental training may indicate the training-induced functional plasticity of the motor activation strategy within the trapezius muscle in individual with bilateral upper-limb congenital transverse deficiency. Marked changes in spatial distribution during the submaximal contraction in the patient after training could be associated with changes of the neural drive to the muscle, which corresponds with specific (unfamiliar for patient) motor task. These findings are relevant to neuromuscular functional rehabilitation in patients with a bilateral upper-limb congenital transverse deficiency especially before and after upper limb transplantation and to development of the EMG based prostheses.

Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line.

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes.

Translational Control in p53 Expression: The Role of 5'-Terminal Region of p53 mRNA.

In this review, the latest research concerning the structure and function of the 5'-terminal region of p53 mRNA was discussed. Special attention was focused on defined structural motifs which are present in this region, as well as their conservation and plausible functional role in translation. It is known that the length of the 5'-terminal region and the structural environment of initiation codons can strongly modulate translation initiation. The ability of this region of p53 mRNA to bind protein factors was also described with special emphasis on general principles that govern, such RNA-protein interactions. The structural alterations within the 5'-terminal region of p53 mRNA and proteins that bind to this region have a strong impact on the rate of mRNA scanning and on translation efficiency in in vitro assays, in selected cell lines, and under stress conditions. Thus, the structural features of the 5'-terminal region of p53 mRNA seem to be very important for translation and for translation regulation mechanisms. Finally, we suggested topics that, in our opinion, should be further explored for better understanding of the mechanisms of the p53 gene expression regulation at the translational level.

Low-Frequency Fatigue Assessed as Double to Single Twitch Ratio after Two Bouts of Eccentric Exercise of the Elbow Flexors.

The aim of this study was to assess low-frequency fatigue as a double to single twitch ratio after repeated eccentric exercise of the elbow flexors. Maximal isometric torque, single and double twitch responses and low-frequency fatigue were assessed on the elbow flexors in 16 untrained male volunteers before, immediately after, 24 and 48 hours following two bouts of eccentric exercise consisted of 30 repetitions of lowering a dumbbell adjusted to ~75% of each individual's maximal isometric torque. Maximal isometric torque and electrically evoked responses decreased significantly in all measurements after the first bout of eccentric exercise (p < 0.05). In measurements performed at 24 and 48 hours after the second bout both maximal voluntary isometric torque and electrically evoked contractions were significantly higher than in measurements performed after the first bout (p < 0.05). Although low-frequency fatigue significantly increased up to 48 hours after each bout of eccentric exercise, its values at 24 and 48 hours after the second bout were significantly lower than at respective time points after the first bout (p < 0.05). Double to single twitch ratio could be used as a sensitive tool in the evaluation of muscle recovery and adaptation to repeated eccentric exercise.

Characterization of RNP Networks of PUM1 and PUM2 Post-Transcriptional Regulators in TCam-2 Cells, a Human Male Germ Cell Model.

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins (RBPs) with wide-ranging roles. They are involved in germ cell development, which has functional implications in development and fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity. To address that problem, first we used RIP-Seq and RNA-Seq approaches, and identified mRNA pools regulated by PUM1 and PUM2 proteins in the TCam-2 cell line, a human male germ cell model. Second, applying global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-interacting putative protein cofactors, most of them involved in RNA processing. Third, combinatorial analysis of RIP and RNA-Seq, mass spectrometry, and RNA motif enrichment analysis revealed that PUM1 and PUM2 form partially varied RNP-regulatory networks (RNA regulons), which indicate different roles in human reproduction and testicular tumorigenesis. Altogether, this work proposes that protein paralogues with very similar and evolutionary highly conserved functional domains may play divergent roles in the cell by combining with different sets of protein cofactors. Our findings highlight the versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

SPIN1 is necessary for normal meiotic progression in mammals. It is overexpressed in human ovarian cancers and some cancer cell lines. Here, we examined the functional significance and regulation of SPIN1 and SPIN3 in the TCam-2 human seminoma cell line. We found that while SPIN1 overexpression reduced apoptosis in these cells, SPIN3 overexpression induced it. Similarly, SPIN1 upregulated and SPIN3 downregulated CYCD1, which is a downstream target of the PI3K/AKT pathway and contributes to apoptosis resistance in cancer cell lines. It appears that SPIN1 is pro-oncogenic and SPIN3 acts as a tumor suppressor in TCam-2 cells. To our knowledge, this is the first report of SPIN3 tumor suppressor activity. However, both SPIN1 and SPIN3 stimulated cell cycle progression. In addition, using luciferase reporters carrying SPIN1 or SPIN3 mRNA 3'UTRs, we found that PUM1 and PUM2 targeted and repressed SPINs. We also found that PUM1 itself strongly stimulated apoptosis and moderately slowed cell cycle progression in TCam-2 cells, suggesting that PUM1, like SPIN3, is a tumor suppressor. Our findings suggest that acting, at least in part, through SPIN1 and SPIN3, PUM proteins contribute to a mechanism promoting normal human male germ cell apoptotic status and thus preventing cancer.

A Case of Wiedemann-Steiner Syndrome Associated with a 46,XY Disorder of Sexual Development and Gonadal Dysgenesis.

We report the case of a female patient suffering from a 46,XY disorder of sexual development (DSD) with complete gonadal dysgenesis and Wiedemann-Steiner Syndrome (WDSTS). The coexistence of these 2 conditions has not yet been reported. Using whole exome sequencing and comparative genome hybridization array, we identified a de novo MLL/KMT2A gene nonsense mutation which explains the WDSTS phenotype. In addition, we discovered novel genetic variants, which could explain the testicular dysgenesis observed in the patient, a maternally inherited 167-kb duplication of DAAM2 and MOCS1 genes and a de novo LRRC33/NRROS gene mutation. These genes, some of which are expressed during mouse gonadal development, could be considered as potentially new candidate genes for DSD.

Twitch mechanical properties after repeated eccentric exercise of the elbow flexors.

The purpose of this study was to assess if the protective adaptation after eccentric exercise affects changes of twitch contractile properties of the biceps brachii muscle. Maximal isometric torque (MVC), twitch contractile properties, muscle soreness, and relaxed elbow angle (RANG) assessments were measured in 12 untrained, right-handed male volunteers (age, 23 ± 2 years; height, 182 ± 5 cm; mass, 75 ± 7 kg) before, immediately after, 48 h, and 120 h following each bout of eccentric exercise that consisted of 30 repetitions of lowering a dumbbell adjusted to 75% of each individual's maximal isometric torque of the right elbow flexors. MVC, peak twitch torque, maximal rate of twitch torque development, maximal rate of relaxation, muscle soreness, and RANG changes were significantly attenuated after the second bout of eccentric exercise when compared with the first bout. In contrast, time to twitch peak torque and half relaxation time did not change significantly after both the first and the second bout. The findings indicate that the mechanisms responsible for rapid adaptation affect some twitch mechanical properties such as peak torque, maximal rate of torque development, and maximal rate of relaxation but not time to peak torque and half relaxation time.

Effects of submaximal eccentric exercise on muscle activity at different elbow joint angles.

Our study aimed to determine whether electrical and mechanical factors contributing to acute or long-term maximal torque reduction and muscle soreness due to submaximal eccentric exercise (ECC) are elbow-joint-angle specific and to what extent the joint angle affects the contribution of antagonist coactivation to this torque reduction. Maximal isometric torque (MIT), muscle soreness assessment, agonist electromechanical activities, and antagonist coactivation during the maximal voluntary contraction (MVC) were measured at elbow joint angles of 60°, 90°, and 150° before ECC, immediately after exercise, and 24, 48, 72, and 120 hr after exercise. ECC causes an immediate decrease in MIT as well as increased antagonist coactivation at three angles. Antagonist coactivation returned to its baseline level at 24 hr regardless of joint angle. The most rapid torque recovery and the highest force level at which pain occurred were found after ECC at a joint angle of 60°. During the recovery period, no mechanomyographical changes were observed when measuring surface mechanomyography changes at three angles, while the electrical activity differed between angles.

Muscle passive stiffness increases less after the second bout of eccentric exercise compared to the first bout.

OBJECTIVES: The purpose of this study was to assess if the protective adaptation after eccentric exercise affects changes in passive stiffness of the biceps brachii muscle. DESIGN: A within-group repeated measures design was used to compare changes in passive muscle stiffness after eccentric exercise between the first and second bouts separated by 2-3 weeks. METHOD: Maximal isometric torque, passive muscle stiffness and soreness were measured on the right elbow flexors in 14 untrained male volunteers before, immediately after, 24, 48 and 120 h following each bout of eccentric exercise that consisted of 30 repetitions of lowering a dumbbell adjusted to 75% of each individual's maximal isometric torque. RESULTS: Maximal isometric torque reduced immediately after the first bout by 24 ± 11% (mean ± SD; P < 0.05) and remained decreased for the next 120 h (~23%). Passive muscle stiffness immediately increased from 223 ± 19 N/m to 254 ± 22 N/m (P < 0.05) and remained higher for 120 h. After the second bout maximal isometric torque decreased 21 ± 13%, and 48 h later recovered to pre-exercise level (P < 0.05). Increase in passive muscle stiffness was attenuated after the second bout (238 ± 17 N/m; P < 0.05). The perceived muscle soreness was lower after the second bout. CONCLUSIONS: Smaller increases in passive muscle stiffness and soreness, and faster maximal isometric torque recovery after the second bout of eccentric exercise could result from adaptation process that occurred after the first bout.