Biology subjects

Paulsen, M. T.

Publications and source records attributed to Paulsen, M. T..

8 recordsLinked to original sources

bioRxiv2024

Isoform and pathway-specific post-transcriptional RNA processing in human cells

Steady-state levels of RNA transcripts are controlled by their rates of synthesis and degradation. Here we used nascent RNA Bru-seq and BruChase-seq to profile RNA dynamics across 16 human cell lines as part of ENCODE4 Deeply Profiled Cell Lines collection. We show that RNA turnover dynamics differ widely between transcripts of different genes and between different classes of RNA. Gene set enrichment analysis (GSEA) revealed that transcripts encoding proteins belonging to the same pathway often show similar turnover dynamics. Furthermore, transcript isoforms show distinct dynamics suggesting that RNA turnover is important in regulating mRNA isoform choice. Finally, splicing across newly made transcripts appears to be cooperative with either all or none type splicing. These data sets generated as part of ENCODE4 illustrate the intricate and coordinated regulation of RNA dynamics in controlling gene expression to allow for the precise coordination of cellular functions.

molecular biology↗

bioRxiv2024

Characterizing nascent transcription patterns of PROMPTs, eRNAs, and readthrough transcripts in the ENCODE4 deeply profiled cell lines

Arising as co-products of canonical gene expression, transcription-associated lincRNAs, such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and readthrough (RT) transcripts, are often regarded as byproducts of transcription, although they may be important for the expression of nearby genes. We identified regions of nascent expression of these lincRNA in 16 human cell lines using Bru-seq techniques, and found distinctly regulated patterns of PROMPT, eRNA, and RT transcription using the diverse biochemical approaches in the ENCODE4 deeply profiled cell lines collection. Transcription of these lincRNAs was influenced by sequence-specific features and the local or 3D chromatin landscape. However, these sequence and chromatin features do not describe the full spectrum of lincRNA expression variability we identify, highlighting the complexity of their regulation. This may suggest that transcription-associated lincRNAs are not merely byproducts, but rather that the transcript itself, or the act of its transcription, is important for genomic function.

molecular biology↗

bioRxiv2021

Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously-encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Accordingly, our findings establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis, but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

neuroscience↗

bioRxiv2020

Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA

Transcription restart after a genotoxic challenge is a fundamental yet poorly understood process. Here, we dissect the interplay between transcription and chromatin restoration after DNA damage by focusing on the human histone chaperone complex HIRA, which is required for transcription recovery post UV. We demonstrate that HIRA is recruited to UV-damaged chromatin via the ubiquitin-dependent segregase VCP to deposit new H3.3 histones. However, this local activity of HIRA is dispensable for transcription recovery. Instead, we reveal a genome-wide function of HIRA in transcription restart that is independent of new H3.3 and not restricted to UV-damaged loci. HIRA coordinates with ASF1B to control transcription restart by two independent pathways: by stabilizing the associated subunit UBN2 and by reducing the expression of the transcription repressor ATF3. Thus, HIRA primes UV-damaged chromatin for transcription restart at least in part by relieving transcription inhibition rather than by depositing new H3.3 as an activating bookmark.

molecular biology↗

bioRxiv2020

KMT2D Links TGF-β Signalling to Non-Canonical Activin Pathway and Regulates Pancreatic Cancer Cell Plasticity

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-{beta} to the activin A pathway. We found that TGF-{beta} upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a non-canonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the pro-tumoral role of KMT2D. These findings reveal a tumor-suppressive role of KMT2D and identify miR-147b and activin A as novel therapeutic targets in pancreatic cancer.

cancer biology↗

bioRxiv2020

Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

The human PUF-family proteins, PUM1 and PUM2, post-transcriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3 UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a rulebook of key contextual features that differentiate functional vs. non-functional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

molecular biology↗

bioRxiv2020

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

cell biology↗

bioRxiv2018

Identification of cis elements for spatio-temporal control of DNA replication

The temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis-elements regulating spatio-temporal control of replication have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide delay in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These \"early replication control elements\" (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis-regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.\n\nHighlightsO_LIcis-elements (ERCEs) regulate large scale chromosome structure and function\nC_LIO_LIMultiple ERCEs cooperatively control domain-wide replication\nC_LIO_LIERCEs harbor prominent active chromatin features and form CTCF-independent loops\nC_LIO_LIERCEs enable genetic dissection of large-scale chromosome structure-function.\nC_LI

molecular biology↗