Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.

DNA damage triggers a complex transcriptional response that involves both activation and repression of gene expression. In this study, we investigated global changes in transcription in response to ionizing irradiation (IR), which induces double-strand breaks in DNA. We used mNET-seq to profile nascent transcripts bound to different phosphorylated forms of the RNA polymerase II (RNA Pol II) C-terminal domain (CTD). We found that IR leads to global transcriptional repression of protein-coding genes, accompanied by an increase in antisense transcripts near promoters, called PROMPTs, transcribed by RNA Pol II phosphorylated on tyrosine 1 (Y1P) residue of the CTD. These Y1P-transcribed PROMPTs are enriched for PRC2 binding sites and associated with RNA Pol II proximal promoter pausing. We show the interaction between Y1P RNA Pol II and PRC2, as well as PRC2 binding to PROMPTs. Inhibition of PROMPTs or depletion of PRC2 leads to loss of transcriptional repression. Our results reveal a novel function of Y1P-dependent PROMPTs in mediating PRC2 recruitment to chromatin and RNA Pol II promoter pausing in response to DNA damage.

Swiss army knives: non-canonical functions of nuclear Drosha and Dicer.

The RNase III enzymes Drosha and Dicer are essential for the production of small non-coding RNAs (ncRNAs). In canonical RNAi, microRNAs (miRNAs) regulate gene expression by post-transcriptional gene silencing. In non-canonical RNAi, nuclear RNAi factors generate small ncRNAs that are essential for transcriptional gene silencing. Recent evidence points to the existence of additional non-canonical nuclear RNAi functions in various organisms, including in genome maintenance and editing, as well as in DNA repair. Drosha and Dicer directly regulate gene expression and RNA metabolism at different stages, such as transcriptional initiation and termination, and the processing of various RNA species, including pre-mRNAs. Furthermore, Dicer isoforms were recently discovered and attributed with roles in apoptosis, development and disease.

Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Observing many researchers using the same data and hypothesis reveals a hidden universe of uncertainty.

This study explores how researchers' analytical choices affect the reliability of scientific findings. Most discussions of reliability problems in science focus on systematic biases. We broaden the lens to emphasize the idiosyncrasy of conscious and unconscious decisions that researchers make during data analysis. We coordinated 161 researchers in 73 research teams and observed their research decisions as they used the same data to independently test the same prominent social science hypothesis: that greater immigration reduces support for social policies among the public. In this typical case of social science research, research teams reported both widely diverging numerical findings and substantive conclusions despite identical start conditions. Researchers' expertise, prior beliefs, and expectations barely predict the wide variation in research outcomes. More than 95% of the total variance in numerical results remains unexplained even after qualitative coding of all identifiable decisions in each team's workflow. This reveals a universe of uncertainty that remains hidden when considering a single study in isolation. The idiosyncratic nature of how researchers' results and conclusions varied is a previously underappreciated explanation for why many scientific hypotheses remain contested. These results call for greater epistemic humility and clarity in reporting scientific findings.

Making it to the Academic Path in a Tracked Education System: The Interplay of Individual Agency and Social Origin in Early Educational Transitions.

Little is known about the role of agency in transitions in tracked education systems or whether it varies by socioeconomic background. This study addressed this gap by estimating structural equation models based on longitudinal data that are representative of the German- and French-speaking parts of Switzerland (N = 1273 individuals, surveyed from age 6 to 18, mean age at wave 1: Mage = 6.54, SDage = 0.50, female = 49%). The findings reveal that agency (captured by study effort and occupational aspirations) and socioeconomic background (measured by parental education and family income) significantly predicted students' transitions to academically demanding tracks in lower- and upper-secondary education. In the transition to upper-secondary education, students with fewer socioeconomic resources benefitted less than their more advantaged peers from ambitious aspirations, but they benefitted more from exerting effort. These findings suggest that both an optimistic forward-looking orientation and the exertion of effort are required to make it to an academic track. Effort may serve as a "substitutive" resource for less socioeconomically advantaged students, whereas ambitious aspirations may enhance the positive effect of family socioeconomic resources on academic educational trajectories. Overall, the evidence from this study calls for greater attention to investigating not only how agency shapes adolescents' educational trajectories and opportunities but also how its role differs across social groups.

Compartment-Specific Proximity Ligation Expands the Toolbox to Assess the Interactome of the Long Non-Coding RNA NEAT1.

The nuclear paraspeckle assembly transcript 1 (NEAT1) locus encodes two long non-coding (lnc)RNA isoforms that are upregulated in many tumours and dynamically expressed in response to stress. NEAT1 transcripts form ribonucleoprotein complexes with numerous RNA-binding proteins (RBPs) to assemble paraspeckles and modulate the localisation and activity of gene regulatory enzymes as well as a subset of messenger (m)RNA transcripts. The investigation of the dynamic composition of NEAT1-associated proteins and mRNAs is critical to understand the function of NEAT1. Interestingly, a growing number of biochemical and genetic tools to assess NEAT1 interactomes has been reported. Here, we discuss the Hybridisation Proximity (HyPro) labeling technique in the context of NEAT1. HyPro labeling is a recently developed method to detect spatially ordered interactions of RNA-containing nuclear compartments in cultured human cells. After introducing NEAT1 and paraspeckles, we describe the advantages of the HyPro technology in the context of other methods to study RNA interactomes, and review the key findings in mapping NEAT1-associated RNA transcripts and protein binding partners. We further discuss the limitations and potential improvements of HyPro labeling, and conclude by delineating its applicability in paraspeckles-related cancer research.

Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks.

DNA is constantly exposed to endogenous and exogenous damage. Various types of DNA repair counteract highly toxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilizes small RNA species, which are produced as long non-coding (nc)RNA precursors and promote recognition of DSBs. However, regulatory principles that control production of such transcripts remain largely elusive. Here we show that the Abelson tyrosine kinase c-Abl/ABL1 causes formation of RNA polymerase II (RNAPII) foci, predominantly phosphorylated at carboxy-terminal domain (CTD) residue Tyr1, at DSBs. CTD Tyr1-phosphorylated RNAPII (CTD Y1P) synthetizes strand-specific, damage-responsive transcripts (DARTs), which trigger formation of double-stranded (ds)RNA intermediates via DNA-RNA hybrid intermediates to promote recruitment of p53-binding protein 1 (53BP1) and Mediator of DNA damage checkpoint 1 (MDC1) to endogenous DSBs. Interference with transcription, c-Abl activity, DNA-RNA hybrid formation or dsRNA processing impairs CTD Y1P foci formation, attenuates DART synthesis and delays recruitment of DDR factors and DSB signalling. Collectively, our data provide novel insight in RNA-dependent DDR by coupling DSB-induced c-Abl activity on RNAPII to generate DARTs for consequent DSB recognition.

Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage.

Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage.

Making it against the odds: How individual and parental co-agency predict educational mobility.

INTRODUCTION: This study examines the role of individual agency and parental co-agency as resource factors enabling educational mobility (university enrolment and degree completion) among first-generation students. METHODS: The study is based on Next Steps, a nationally representative cohort of UK students. Path models were run, linking different dimensions of agency assessed at age 13/14 to educational attainment by age 25/26, controlling for academic attainment and socio-demographic factors. RESULTS: Educational mobility was predicted by student's expectation to go to university, their expectation of success, and school engagement during secondary school. In addition, parental co-agency played a significant role - highlighting the importance of parents in supporting upward educational mobility of their children. CONCLUSIONS: Multiple dimensions of agency are necessary for disadvantaged students to achieve academically. To support first-generation students, schools need to provide opportunities for them to become engaged in education, to experience mastery and to develop realistic expectations of success.

Self-esteem and self-efficacy in the status attainment process and the multigenerational transmission of advantage.

Despite considerable evidence of the importance of self-esteem and self-efficacy for agentic, goal-oriented behavior, little attention has been directed to these psychological dimensions in the status attainment literature. The present research uses data from the longitudinal, three-generation Youth Development Study (N = 422 three-generation triads) to examine the extent to which adolescent self-esteem and economic self-efficacy affect adult educational and income attainment, and whether these psychological resources are transmitted from one generation to the next, accumulating advantage across generations. We present evidence indicating that both self-esteem and economic self-efficacy are implicated in the attainment process. Adolescent economic self-efficacy had a direct positive effect on adult educational attainment and an indirect effect through educational plans. The influence of self-esteem on adult educational attainment was entirely indirect, through school achievement. We also find evidence that economic self-efficacy was transmitted from parents to children. We conclude that future research should more broadly consider psychological resources in attainment processes from a longitudinal multigenerational perspective.

Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation.

Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice.

The Role of Perceived Stress and Self-Efficacy in Young People's Life Satisfaction: A Longitudinal Study.

Life satisfaction is an important indicator of successful development. However, adolescents' life satisfaction tends to be relatively unsteady, and environmental influences play a critical role in shaping life satisfaction among adolescents in the transition to young adulthood. Given the paramount importance that education plays in adolescents' lives, adolescents' life satisfaction may vary as a function of school-related stress experience. At the same time, coping resources may help reduce adverse effects of stress on life satisfaction. With this in mind, we examined whether, and to what extent, perceived stress in education and general self-efficacy (a resource that facilitates coping) affect the life satisfaction of adolescents in transition to young adulthood. We distinguished between baseline levels of stress and self-efficacy and within-person change in stress and self-efficacy to determine whether life satisfaction is sensitive to fluctuations in stress and self-efficacy when person-specific levels of stress and self-efficacy are taken into account. Estimating growth curve models on data from a panel study on the life trajectories of compulsory-school leavers (n = 5126, 55.3 % female), we found that baseline levels of stress and self-efficacy, as well as within-person change in stress and self-efficacy, affected adolescents' life satisfaction. Moreover, our results showed that baseline self-efficacy mitigated the negative effect of baseline stress on life satisfaction. These findings improve our understanding of two major psychological determinants of adolescents' life satisfaction and extend our knowledge of life satisfaction trajectories during the transition to young adulthood.

DEAD-box helicase DDX27 regulates 3' end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex.

PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3' end formation of 47S rRNA independently of the PeBoW-complex.

Ctk1 function is necessary for full translation initiation activity in Saccharomyces cerevisiae.

Translation is a fundamental and highly regulated cellular process. Previously, we reported that the kinase and transcription elongation factor Ctk1 increases fidelity during translation elongation in Saccharomyces cerevisiae. Here, we show that loss of Ctk1 function also affects the initiation step of translation. Translation active extracts from Ctk1-depleted cells show impaired translation activity of capped mRNA, but not mRNA reporters containing the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Furthermore, the formation of 80S initiation complexes is decreased, which is probably due to reduced subunit joining. In addition, we determined the changes in the phosphorylation pattern of a ribosome enriched fraction after depletion of Ctk1. Thus, we provide a catalogue of phosphoproteomic changes dependent on Ctk1. Taken together, our data suggest a stimulatory function of Ctk1 in 80S formation during translation initiation.

Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution.

RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, had so far remained unresolved. Here, we show that ultrashort 4sU-tagging not only provides snapshot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length, and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression.

Cyclin-dependent kinase 9 links RNA polymerase II transcription to processing of ribosomal RNA.

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

Mental health and educational attainment: How developmental stage matters.

Developmental science suggests that the consequences of mental health problems for life-course outcomes may depend on the timing of their onset. This study investigated the extent to which mental health predicted educational attainment at ages 17, 20, and 25 and whether gender moderated the links between mental health and educational attainment. It used data from Next Steps, a nationally representative panel survey of individuals born in 1989/1990 in England (N = 15,594, 48% female, 33% ethnic minority). The findings suggest that differences in mental health were more consequential for educational attainment during adolescence than in young adulthood. On average, girls attained higher levels of education than boys, but gender did not moderate the role that mental health played for educational attainment. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Nono induces Gadd45b to mediate DNA repair.

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.

Mental health, gender, and higher education attainment.

We compared the mental health of higher education students with that of nonstudents. Moreover, we examined whether the mental health of students predicts their probability of obtaining a higher education degree, and whether the extent to which mental health affects educational attainment varies by gender. Drawing on a risk and resilience framework, we considered five facets of mental health that may be implicated in distinct ways in the educational attainment process: positive attitude towards life, self-esteem, self-efficacy, negative affectivity, and perceived stress. We used data from a nationally representative panel study from Switzerland (Nstudents = 2070, 42.8% male; Nnonstudents = 3755, 45.9% male). The findings suggest that overall, the mental health of higher education students was relatively similar to that of nonstudents, although students exhibited slightly higher self-esteem, slightly weaker self-efficacy, greater negative affectivity, and higher levels of perceived stress. The effects of different facets of mental health on higher education degree attainment were mostly statistically and/or practically insignificant. However, positive attitudes towards life had a substantial positive effect on the probability of being awarded a higher education degree. Mental health was equally important for male and female students' educational attainment. Supplementary Information: The online version of this article (10.1007/s11618-023-01187-3) contains supplementary material, which is available to authorized users.

Functional ribosome biogenesis is a prerequisite for p53 destabilization: impact of chemotherapy on nucleolar functions and RNA metabolism.

The production and processing of ribosomal RNA is a complex and well-coordinated nucleolar process for ribosome biogenesis. Progress in understanding nucleolar structure and function has lead to the unexpected discovery of the nucleolus as a highly sensitive sensor of cellular stress and an important regulator of the tumor suppressor p53. Inhibition of ribosomal RNA metabolism has been shown to activate a signaling pathway for p53 induction. This review elucidates the potential of classical and recently developed chemotherapeutic drugs to stabilize p53 by inhibiting nucleolar functions.