Biology subjects

Stepien, A.

Publications and source records attributed to Stepien, A..

8 recordsLinked to original sources

bioRxiv2025

Proximity of pre-mRNA 3' end processing and transcription termination predicts enhanced gene expression

Two key factors required for pre-mRNA 3 end cleavage and polyadenylation, CPSF73 and PCF11, exhibit oncogenic properties: their elevated expression is associated with poor cancer patient prognosis. However, both proteins are also transcription termination factors, and it is unclear which of the processes they promote might contribute to carcinogenesis. Here, we employ a cellular model of colorectal cancer (CRC) progression and find that cells from primary tumor are addicted to high levels of CPSF73 and PCF11, while metastatic cells become less sensitive to their levels. We find no association between alternative polyadenylation (APA) and cell dependence on CPSF73 and PCF11, and no impact of their downregulation on transcription-replication collisions. Instead, we uncover an uncoupling of changes in 3 cleavage and termination during CRC progression: primary tumor-derived cells display a global shift to more proximal termination, yet a tendency for distal APA, compared to normal cells. Metastatic cells display partial reversion toward termination patterns observed in normal cells, and opposite tendency favoring proximal APA. This prompts us to measure the distance between the site of 3 cleavage and transcription termination for active protein-coding genes and find it almost halved in cells from primary tumor compared to normal cells. Interestingly, this distance becomes critically short for oncogenes. Closer proximity of 3 cleavage to termination correlates with higher gene expression, both across genes within a cell line and when distance and expression change in parallel. This uncovers a new relationship of transcription termination with gene expression regulation. SIGNIFICANCE STATEMENTAt the 3 end of genes two processes occur, which are often treated as one: pre-mRNA 3 processing and transcription termination. In fact, they are promoted by a common set of protein factors, with oncogenic properties. Here, we employ a cellular model of colorectal cancer (CRC) progression, to study changes in both pre-mRNA 3 processing and transcription termination. Unexpectedly, we uncover that those changes occur in opposite directions: mRNA tends to be lengthened, while termination is accelerated in cells from primary tumors. We show that the cleavage-termination distance is strongly reduced in these cells compared to normal and metastatic cells, and further determine that cleavage-termination proximity correlates with enhanced gene expression. This uncovers a new layer of gene expression regulation.

molecular biology↗

bioRxiv2025

SETD2 methyltransferase activity aids gene definition by promoting correct transcription initiation and termination

SETD2 is a methyltransferase responsible for depositing histone H3 lysine 36 trimethylation (H3K36me3). Loss of its enzymatic activity occurs in some cancers, including renal cell carcinoma (RCC). In RCC, SETD2 mutations have been linked to delayed transcription termination, but not explored in depth. Here, using nascent transcriptomics in SETD2 knockout and patient-derived cells, we reveal a dichotomy in SETD2 functions depending on the affected protein-coding gene. Majority of genes, named class I, are dependent on SETD2 function for transcription initiation, yet terminate transcription in the usual locations. In contrast, for class II genes, corresponding to 15-25% of active protein-coding genes, transcription initiation is robust in absence of SETD2 activity, however widespread transcriptional readthrough occurs. Defective termination following SETD2 loss/mutation is associated with increased cryptic transcription initiation and impaired 3' pre-mRNA cleavage. Additionally, alternative polyadenylation upon SETD2 activity loss is highly cell type specific, and unrelated to transcription readthrough. In summary, we demonstrate that methyltransferase activity of SETD2 regulates transcriptional integrity by stimulating proper initiation, preventing cryptic initiation and promoting efficient 3' end processing.

molecular biology↗

bioRxiv2025

BRG1 targeting overcomes ABCC-based multidrug resistance induced by paclitaxel

Multidrug resistance of cancer cells is attributed to drug-induced alteration of numerous intracellular processes. Using clinically relevant models of triple-negative breast and non-small lung cancer cells we previously showed that these cells respond to repeated paclitaxel exposure by inter alia lysosome enrichment in ABCC3, ABCC5 and ABCC10, which contribute to drug sequestration in these organelles and reduced drug cytotoxicity. In this study we provide experimental evidence that transcription of above mentioned ABCC genes is enabled by BRG1-based SWI/SNF chromatin remodeling complex. Pharmacological inhibition of SWI/SNF with PFI3 or ACBI1, the PROTAC degrader of SMARCA2/4, substantially decline transcription of ABCC3, ABCC5 and ABCC10. Similar effect is caused by transient silencing of SMARCA4 (BRG1), but not SMARCA2 (BRM). The deficiency of BRG1 led to extralysosomal distribution of anticancer drugs, their deeper penetration of spheroids and substantial increase in drug cytotoxicity. Interestingly, in BRG1 deficient cell line paclitaxel triggered mutations, which reverted BRG1 truncating deletion in SMARCA4, thereby restoring SWI/SNF ATPase expression in paclitaxel-resistant cells and increasing transcription of ABCC. Acquisition of drug resistance was associated with BRG1 redistribution in the genome, de novo occurrence at the promoters of genes functionally linked to endo-lysosomal system and stronger co-occurrence with EP300. Our study indicates possible target - SWI/SNF complex for anticancer combinatorial interventions in paclitaxel-induced multidrug resistant phenotypes.

cancer biology↗

bioRxiv2023

Novel function of U7 snRNA in the repression of HERV1/LTR12s and lincRNAs in human cells

U7 snRNA is part of U7 snRNP, a complex required for the 3end processing of replication-dependent histone pre-mRNAs in the S phase of the cell cycle. During this maturation event, the 5 region of U7 snRNA hybridizes with the highly complementary sequence present in the 3UTR of histone pre-mRNAs, called histone downstream element, HDE. This base-pair interaction triggers subsequent reactions that eventually result in cleavage and release of mature histone transcripts. Intriguingly, U7 snRNP is constitutively expressed throughout the cell cycle and in nondividing cells, suggesting another function of U7 snRNA/snRNP in cells. Here, we show that several human endogenous retroviruses (HERVs) are significantly upregulated in HEK293T cells with U7 snRNA knockdown. They predominantly belong to the LTR12 class. Interestingly, some of them are located within long intergenic noncoding RNAs (lincRNAs), which in turn are upregulated in U7 snRNA knockdown cells as well. Significantly, both these HERV1/LTR12s and lincRNAs contain two or more sequence motifs that perfectly match the 5 end of U7 snRNA, which we called HDE-like motifs. We confirmed that mutations within the HDE-like motifs abrogate U7 snRNA regulatory function and stimulate the expression of selected lincRNAs. Furthermore, we demonstrate that U7 snRNA inhibits HERV1/LTR12 and lincRNA expression at the transcription level. We propose a mechanism in which U7 snRNA hampers binding/activity of NF-Y transcription factor to CCAAT motifs that are frequently found in LTRs as well as in a close proximity to HDE-like motifs. The expression of many HERV1/LTR12s and lincRNAs regulated by U7 snRNA seems to be tissue specific, therefore, we suggest that U7 snRNA plays a protective role in keeping deleterious genetic elements in silence in selected types of cells.

molecular biology↗

bioRxiv2022

Chromatin-associated microprocessor assembly is regulated by PRP40, the U1 snRNP auxiliary protein

Cotranscriptional processing of RNA polymerase II-generated primary transcripts is a well-documented phenomenon. We recently showed that in plants, miRNA biogenesis is also a cotranscriptional event. Here, we report that Arabidopsis PRP40, the U1 snRNP auxiliary protein, positively regulates the recruitment of SE, the core component of the plant microprocessor, to miRNA genes. The association of DCL1, the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNA polymerase II accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.

molecular biology↗

bioRxiv2018

FUS controls the processing of snoRNAs into smaller RNA fragments that can regulate gene expression

FUS is a multifunctional protein involved in many steps of RNA metabolism, including transcription, splicing, miRNA processing and replication-dependent histone gene expression. In this paper, we show for the first time that FUS binds and negatively regulates the levels of a subset of snoRNAs in cells. Scanning of available human small RNA databases revealed the existence of smaller RNA fragments that can be processed from FUS-dependent snoRNAs. Therefore, we suggest that FUS mediates the biogenesis of snoRNA-derived small RNAs, called sdRNAs. Further in silico approaches enabled us to predict putative targets of selected FUS-dependent sdRNAs. Our results indicate that sdRNAs may bind to different regions of target mRNAs as well as to noncoding transcripts and influence the posttranscriptional level or translation of these targets. SIGNIFICANCE STATEMENTRNA metabolism is orchestrated by a complex network of RNA-protein interactions and involves various classes of RNA molecules. Small nucleolar RNAs (snoRNAs) are commonly considered essential components of the ribosome biogenesis pathway. However, recent studies have revealed that snoRNAs can also be fragmented into small entities called snoRNA-derived RNAs (sdRNAs), which have been linked to multiple cancer types and thus may serve as next-generation prognostic or diagnostic biomarkers. In this paper, a multifunctional protein, FUS, was shown to be involved in the biogenesis of snoRNA-derived fragments. Furthermore, we combined bioinformatic analyses with complementary experimental approaches to elucidate the role of FUS-dependent sdRNAs in gene expression regulation. Our findings reveal the considerable regulatory potential of this new class of small noncoding RNAs.

molecular biology↗

bioRxiv2017

Risk factor profile of different clinical manifestations of cerebral small vessel disease - data from SHEF-CSVD Study.

BACKGROUNDLittle is known of the mechanisms of cerebral small vessel disease (CSVD). Both atherosclerosis or non-atherosclerotic diffuse arteriopathy are involved.\n\nMETHODSA single-center, prospective, case-control study was performed in consecutive patients with different CSVD manifestations. The study group consisted of 205 patients: 52 with lacunar stroke (LS), 20 with subcortical hemorrhagic stroke (HS), 50 with vascular dementia (VaD), 28 with vascular parkinsonism (VaP) and 55 controls (CG) free of cerebrovascular disease but with high vascular risk.\n\nRESULTSPatients with CSVD had significantly higher prevalence of vascular risk factors including hypertension, diabetes mellitus, polymetabolic syndrome and chronic kidney disease. Patients with CSVD had also significantly higher fasting blood glucose, homocysteine, fibrinogen, systolic blood pressure, IMT values and lower eGFR, albumin and HDL levels. After adjustment for age and sex, low eGFR, albumin and high levels of uric acid and fibrinogen were associated with all CSVD groups, elevated fasting glucose was related to LS and HS. In the multivariate analysiss, the independent predictors for CSVD were female sex, low albumin, high fibrinogen, fasting glucose and uric acid. Patients with LS had significantly higher IMT values comparing to other CSVD groups, patients with VaP had a trend towards higher homocysteine levels.\n\nCONCLUSIONRisk factor profile for CSVD as a whole differs from subjects with proatherogenic profile without history of cerebrovascular disease. Our results support the concept that CSVD is not homogeneous, and that unique risk factors profiles exist for different clinical manifestations of the disease.

neuroscience↗

bioRxiv2017

Risk of vascular events or death in different manifestations of cerebral small vessel disease: a 2-year follow-up study with a control group

Background and PurposeNatural course of cerebral small vessel disease (CSVD) has not yet been thoroughly studied. The aim of the single center study was to establish risk of vascular events or death in different manifestations of CSVD.\n\nMethods150 consecutive, functionally independent patients with marked MRI features of CSVD and with recent lacunar stroke (n=52, LS), 20 with deep hemorrhagic stroke (HS), 28 with vascular parkinsonism (VaP), 50 with vascular dementia (VaD) and 55 controls (CG) with high atherothrombotic risk free of cerebrovascular events were prospectively recruited and followed for 24 months.\n\nResultsMean age and sex distribution were similar in CSVD and CG but patients with CSVD were less likely to have CAD (19% vs 40%, p=0,02) and tended to have higher prevalence of diabetes (54% vs 37%, p=0,11). The risk of vascular events or death was increased in any patients with moderate to severe white matter lesions at baseline MRI (HR 2,0; 95%CI 0,85-7,2), in CSVD (4,56; 95%CI 1,3-14,9) vs CG, regardless of its clinical manifestation: LS or HS (HR 4,70; 95%CI 1,3-16,2) and VaD or VaP (HR 4,59; 95%CI 1,3-15,7).\n\nConclusionsPatients with symptomatic CSVD regardless of the clinical (acute or chronic) manifestation had more than fourfold the risk of vascular events or death in 24 months of observation compared with controls with high atherothrombotic risk free of cerebrovascular events.

neuroscience↗