Long non-coding RNA generated from CDKN1A gene by alternative polyadenylation regulates p21 expression during DNA damage response.

Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during the DNA damage response (DDR). Here, we show that a DDR-activated APA event occurs in the first intron of CDKN1A, inducing an alternate last exon-containing lncRNA. We named this lncRNA SPUD (Selective Polyadenylation Upon DNA Damage). SPUD localizes to polysomes in the cytoplasm and is detectable as multiple isoforms in available high-throughput studies. SPUD has low abundance compared to the CDKN1A full-length isoform under non-stress conditions, and SPUD is induced in cancer and normal cells under a variety of DNA damaging conditions in part through p53. The RNA binding protein HuR binds to and promotes the stability of SPUD precursor RNA. SPUD induction increases p21 protein, but not mRNA levels, affecting p21 functions in cell-cycle, CDK2 expression and cell growth. Like CDKN1A full-length isoform, SPUD can bind two competitive p21 translational regulators, the inhibitor calreticulin and the activator CUGBP1; SPUD alters their association with CDKN1A full-length in a DDR-dependent manner, promoting CDKN1A translation. Together, these results show a new regulatory mechanism by which a lncRNA controls p21 expression post-transcriptionally, highlighting lncRNA relevance in DDR progression and cell-cycle.

Long Non-Coding RNA Generated from CDKN1A Gene by Alternative Polyadenylation Regulates p21 Expression during DNA Damage Response.

Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during the DNA damage response (DDR). Here, we show that a DNA damage activated APA event occurs in the first intron of CDKN1A , inducing an alternate last exon (ALE)-containing lncRNA. We named this lncRNA SPUD (Selective Polyadenylation Upon Damage). SPUD localizes to polysomes in the cytoplasm and is detectable as multiple isoforms in available high throughput studies. SPUD has low abundance compared to the CDKN1A full-length isoform and is induced in cancer and normal cells under a variety of DNA damaging conditions in part through p53 transcriptional activation. RNA binding protein (RBP) HuR and the transcriptional repressor CTCF regulate SPUD levels. SPUD induction increases p21 protein, but not CDKN1A full-length levels, affecting p21 functions in cell-cycle, CDK2 expression, and cell viability. Like CDKN1A full-length isoform, SPUD can bind two competitive p21 translational regulators, the inhibitor calreticulin and the activator CUGBP1; SPUD can change their association with CDKN1A full-length in a DDR-dependent manner. Together, these results show a new regulatory mechanism by which a lncRNA controls p21 expression post-transcriptionally, highlighting lncRNA relevance in DDR progression and cellcycle.

Epigenome-Wide Study Identifies Epigenetic Outliers in Normal Mucosa of Patients with Colorectal Cancer.

Nongenetic predisposition to colorectal cancer continues to be difficult to measure precisely, hampering efforts in targeted prevention and screening. Epigenetic changes in the normal mucosa of patients with colorectal cancer can serve as a tool in predicting colorectal cancer outcomes. We identified epigenetic changes affecting the normal mucosa of patients with colorectal cancer. DNA methylation profiling on normal colon mucosa from 77 patients with colorectal cancer and 68 controls identified a distinct subgroup of normally-appearing mucosa with markedly disrupted DNA methylation at a large number of CpGs, termed as "Outlier Methylation Phenotype" (OMP) and are present in 15 of 77 patients with cancer versus 0 of 68 controls (P < 0.001). Similar findings were also seen in publicly available datasets. Comparison of normal colon mucosa transcription profiles of patients with OMP cancer with those of patients with non-OMP cancer indicates genes whose promoters are hypermethylated in the OMP patients are also transcriptionally downregulated, and that many of the genes most affected are involved in interactions between epithelial cells, the mucus layer, and the microbiome. Analysis of 16S rRNA profiles suggests that normal colon mucosa of OMPs are enriched in bacterial genera associated with colorectal cancer risk, advanced tumor stage, chronic intestinal inflammation, malignant transformation, nosocomial infections, and KRAS mutations. In conclusion, our study identifies an epigenetically distinct OMP group in the normal mucosa of patients with colorectal cancer that is characterized by a disrupted methylome, altered gene expression, and microbial dysbiosis. Prospective studies are needed to determine whether OMP could serve as a biomarker for an elevated epigenetic risk for colorectal cancer development. PREVENTION RELEVANCE: Our study identifies an epigenetically distinct OMP group in the normal mucosa of patients with colorectal cancer that is characterized by a disrupted methylome, altered gene expression, and microbial dysbiosis. Identification of OMPs in healthy controls and patients with colorectal cancer will lead to prevention and better prognosis, respectively.

Nuclear Tau, p53 and Pin1 Regulate PARN-Mediated Deadenylation and Gene Expression.

While nuclear tau plays a role in DNA damage response (DDR) and chromosome relaxation, the mechanisms behind these functions are not fully understood. Here, we show that tau forms complex(es) with factors involved in nuclear mRNA processing such as tumor suppressor p53 and poly(A)-specific ribonuclease (PARN) deadenylase. Tau induces PARN activity in different cellular models during DDR, and this activation is further increased by p53 and inhibited by tau phosphorylation at residues implicated in neurological disorders. Tau's binding factor Pin1, a mitotic regulator overexpressed in cancer and depleted in Alzheimer's disease (AD), also plays a role in the activation of nuclear deadenylation. Tau, Pin1 and PARN target the expression of mRNAs deregulated in AD and/or cancer. Our findings identify novel biological roles of tau and toxic effects of hyperphosphorylated-tau. We propose a model in which factors involved in cancer and AD regulate gene expression by interactions with the mRNA processing machinery, affecting the transcriptome and suggesting insights into alternative mechanisms for the initiation and/or developments of these diseases.

Hyperphosphorylation of Tau Associates With Changes in Its Function Beyond Microtubule Stability.

Tau is a neuronal microtubule associated protein whose main biological functions are to promote microtubule self-assembly by tubulin and to stabilize those already formed. Tau also plays an important role as an axonal microtubule protein. Tau is an amazing protein that plays a key role in cognitive processes, however, deposits of abnormal forms of tau are associated with several neurodegenerative diseases, including Alzheimer disease (AD), the most prevalent, and Chronic Traumatic Encephalopathy (CTE) and Traumatic Brain Injury (TBI), the most recently associated to abnormal tau. Tau post-translational modifications (PTMs) are responsible for its gain of toxic function. Alonso et al. (1996) were the first to show that the pathological tau isolated from AD brains has prion-like properties and can transfer its toxic function to the normal molecule. Furthermore, we reported that the pathological changes are associated with tau phosphorylation at Ser199 and 262 and Thr212 and 231. This pathological version of tau induces subcellular mislocalization in cultured cells and neurons, and translocates into the nucleus or accumulated in the perinuclear region of cells. We have generated a transgenic mouse model that expresses pathological human tau (PH-Tau) in neurons at two different concentrations (4% and 14% of the total endogenous tau). In this model, PH-Tau causes cognitive decline by at least two different mechanisms: one that involves the cytoskeleton with axonal disruption (at high concentration), and another in which the apparent neuronal morphology is not grossly affected, but the synaptic terminals are altered (at lower concentration). We will discuss the putative involvement of tau in proteostasis under these conditions. Understanding tau's biological activity on and off the microtubules will help shed light to the mechanism of neurodegeneration and of normal neuronal function.

mRNA Processing Factor CstF-50 and Ubiquitin Escort Factor p97 Are BRCA1/BARD1 Cofactors Involved in Chromatin Remodeling during the DNA Damage Response.

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR.

Intronic cleavage and polyadenylation regulates gene expression during DNA damage response through U1 snRNA.

The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3' untranslated region is substantial, leading to both shortening and lengthening of 3' untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5' end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis.

PARN deadenylase is involved in miRNA-dependent degradation of TP53 mRNA in mammalian cells.

mRNA deadenylation is under the control of cis-acting regulatory elements, which include AU-rich elements (AREs) and microRNA (miRNA) targeting sites, within the 3' untranslated region (3' UTRs) of eukaryotic mRNAs. Deadenylases promote miRNA-induced mRNA decay through their interaction with miRNA-induced silencing complex (miRISC). However, the role of poly(A) specific ribonuclease (PARN) deadenylase in miRNA-dependent mRNA degradation has not been elucidated. Here, we present evidence that not only ARE- but also miRNA-mediated pathways are involved in PARN-mediated regulation of the steady state levels of TP53 mRNA, which encodes the tumor suppressor p53. Supporting this, Argonaute-2 (Ago-2), the core component of miRISC, can coexist in complexes with PARN resulting in the activation of its deadenylase activity. PARN regulates TP53 mRNA stability through not only an ARE but also an adjacent miR-504/miR-125b-targeting site in the 3' UTR. More importantly, we found that miR-125b-loaded miRISC contributes to the specific recruitment of PARN to TP53 mRNA, and that can be reverted by the ARE-binding protein HuR. Together, our studies provide new insights into the role of PARN in miRNA-dependent control of mRNA decay and into the mechanisms behind the regulation of p53 expression.

Deadenylation and its regulation in eukaryotic cells.

Messenger RNA deadenylation is a process that allows rapid regulation of gene expression in response to different cellular conditions. The change of the mRNA poly(A) tail length by the activation of deadenylation might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. Activation of deadenylation processes are highly regulated and associated with different cellular conditions such as cancer, development, mRNA surveillance, DNA damage response, and cell differentiation. In the last few years, new technologies for studying deadenylation have been developed. Here we overview concepts related to deadenylation and its regulation in eukaryotic cells. We also describe some of the most commonly used protocols to study deadenylation in eukaryotic cells.

To polyadenylate or to deadenylate: that is the question.

mRNA polyadenylation and deadenylation are important processes that allow rapid regulation of gene expression in response to different cellular conditions. Almost all eukaryotic mRNA precursors undergo a co-transcriptional cleavage followed by polyadenylation at the 3' end. After the signals are selected, polyadenylation occurs to full extent, suggesting that this first round of polyadenylation is a default modification for most mRNAs. However, the length of these poly(A) tails changes by the activation of deadenylation, which might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. The mechanisms behind deadenylation activation are highly regulated and associated with cellular conditions such as development, mRNA surveillance, DNA damage response, cell differentiation and cancer. After deadenylation, depending on the cellular response, some mRNAs might undergo an extension of the poly(A) tail or degradation. The polyadenylation/deadenylation machinery itself, miRNAs, or RNA binding factors are involved in the regulation of polyadenylation/deadenylation. Here, we review the mechanistic connections between polyadenylation and deadenylation and how the two processes are regulated in different cellular conditions. It is our conviction that further studies of the interplay between polyadenylation and deadenylation will provide critical information required for a mechanistic understanding of several diseases, including cancer development.

Nuclear deadenylation/polyadenylation factors regulate 3' processing in response to DNA damage.

We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.

Connections between 3'-end processing and DNA damage response.

The cellular DNA damage response (DDR) involves changes in the functional and structural properties of a number of nuclear proteins, resulting in a coordinated control of gene expression and DNA repair. This response includes functional interactions of the DNA repair, transcription, and RNA processing machineries. Following DNA damage, cellular levels of polyadenylated transcripts are transiently decreased and normal recovery depends on transcription-coupled repair (TCR). In addition, DNA damage has gene-specific effects regulating the mRNA levels of factors involved in the DDR itself at different times after the damage. The 3'-end processing machinery, which is important in the regulation of mRNA stability, is involved in these general and gene-specific responses to DNA damage. The role of 3'-end processing in DDR supports the idea that the steady-state levels of different mRNAs change upon DNA-damaging conditions as a result of regulation of not only their biosynthesis but also their turnover. Here, we review the mechanistic connections between 3'-end processing and DDR, and discuss the implications of deregulation of this important step of mRNA maturation in the cellular recovery after DNA-damaging treatment. The relevance of these functional connections is illustrated by the increasing number of reports on this relatively unexplored field.

The 3' processing factor CstF functions in the DNA repair response.

Following DNA damage, mRNA levels decrease, reflecting a coordinated interaction of the DNA repair, transcription and RNA processing machineries. In this study, we provide evidence that transcription and polyadenylation of mRNA precursors are both affected in vivo by UV treatment. We next show that the polyadenylation factor CstF, plays a direct role in the DNA damage response. Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNA polymerase II (RNAP II), and defects in repair of DNA damage. Furthermore, we show that CstF, RNAP II and BARD1 are all found at sites of repaired DNA. Our results indicate that CstF plays an active role in the response to DNA damage, providing a link between transcription-coupled RNA processing and DNA repair.

Multiple properties of the splicing repressor SRp38 distinguish it from typical SR proteins.

The SR protein SRp38 is a general splicing repressor that is activated by dephosphorylation during mitosis and in response to heat shock. Here we describe experiments that provide insights into the mechanism by which SRp38 functions in splicing repression. We first show that SRp38 redistributes and colocalizes with snRNPs, but not with a typical SR protein, SC35, during mitosis and following heat shock. Supporting the functional significance of this association, a micrococcal nuclease-sensitive component, i.e., an snRNP(s), completely rescued heat shock-induced splicing repression in vitro, and purified U1 snRNP did so partially. SRp38 contains an N-terminal RNA binding domain (RBD) and a C-terminal RS domain composed of two subdomains (RS1 and RS2 domains). Unexpectedly, an RS1 deletion mutant derivative specifically inhibited the second step of splicing, while an RS2 deletion mutant retained significant dephosphorylation-dependent repression activity. Using chimeric SRp38/SC35 proteins, we show that SC35-RBD/SRp38-RS can function as a general splicing activator and that the dephosphorylated version can act as a strong splicing repressor. SRp38-RBD/SC35-RS, however, was essentially inactive in these assays. Together, our results help to define the unusual features of SRp38 that distinguish it from other SR proteins.

BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II.

Mammalian cells exhibit a complex response to DNA damage. The tumor suppressor BRCA1 and associated protein BARD1 are thought to play an important role in this response, and our previous work demonstrated that this includes transient inhibition of the pre-mRNA 3' processing machinery. Here we provide evidence that this inhibition involves proteasomal degradation of a component necessary for processing, RNA polymerase II (RNAP II). We further show that RNAP IIO, the elongating form of the enzyme, is a specific in vitro target of the BRCA1/BARD1 ubiquitin ligase activity. Significantly, siRNA-mediated knockdown of BRCA1 and BARD1 resulted in stabilization of RNAP II after DNA damage. In addition, inhibition of 3' cleavage induced by DNA damage was reverted in extracts of BRCA1-, BARD1-, or BRCA1/BARD1-depleted cells. We also describe corresponding changes in the nuclear localization and/or accumulation of these factors following DNA damage. Our results support a model in which a BRCA1/BARD1-containing complex functions to initiate degradation of stalled RNAP IIO, inhibiting the coupled transcription-RNA processing machinery and facilitating repair.

Autoubiquitination of the BRCA1*BARD1 RING ubiquitin ligase.

The RING finger of BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another RING-containing protein, BARD1, and is required for the function of this tumor suppressor protein in protecting genomic integrity. Here, we report that co-expression of BRCA1-(1-639) and BARD1 in bacteria can assemble a potent ubiquitin ligase activity. Purified BRCA1-(1-639)*BARD1 stimulated the Ubc5c-mediated monoubiquitination of histone H2A/H2AX in vitro, suggesting a possible role for BRCA1*BARD1 in modifying chromatin structure. Moreover, the truncated BRCA1*BARD1 complex exhibited efficient autoubiquitination activity in vitro capable of assembling non-lysine 48-linked polyubiquitin chains on both BRCA1-(1-639) and BARD1. When co-expressed in cells by transient transfection, the recombinant BRCA1-(1-300).BARD1 complex was found to be associated with polyubiquitin chains, suggesting that BRCA1-(1-300)*BARD1 was ubiquitinated in vivo as well. These results raise the possibility that BRCA1*BARD1 acts to assemble non-lysine 48-linked polyubiquitin chains that may serve as part of a signaling platform required for coordinating DNA repair-related events.