Frida E. Kleiman

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.

Poly(A) binding proteins: are they all created equal?

The PABP family of proteins were originally thought of as a simple shield for the mRNA poly(A) tail. Years of research have shown that PABPs interact not only with the poly(A) tail, but also with specific sequences in the mRNA, having a general and specific role on the metabolism of different mRNAs. The complexity of PABPs function is increased by the interactions of PABPs with factors involved in different cellular functions. PABPs participate in all the metabolic pathways of the mRNA: polyadenylation/deadenylation, mRNA export, mRNA surveillance, translation, mRNA degradation, microRNA‐associated regulation, and regulation of expression during development. In this review, we update information on the roles of PABPs and emerging data on the specific interactions of PABP homologs. Specific functions of individual members of PABPC family in development and viral infection are beginning to be elucidated. However, the interactions are complex and recent evidence for exchange of nuclear and cytoplasmic forms of the proteins, as well as post‐translational modifications, emphasize the possibilities for fine‐tuning the PABP metabolic network. WIREs RNA 2013, 4:167–179. doi: 10.1002/wrna.1151

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.

DNA Damage–Induced BARD1 Phosphorylation Is Critical for the Inhibition of Messenger RNA Processing by BRCA1/BARD1 Complex

BRCA1-associated RING domain protein BARD1, along with its heterodimeric partner BRCA1, plays important roles in cellular response to DNA damage. Immediate cellular response to genotoxic stress is mediated by a family of phosphoinositide 3-kinase-related protein kinases, such as ataxia-telangiectasia mutated (ATM), ATM and Rad3-related, and DNA-dependent protein kinase. ATM-mediated phosphorylation of BRCA1 enhances the DNA damage checkpoint functions of BRCA1, but how BARD1 is regulated during DNA damage signaling has not been examined. Here, we report that BARD1 undergoes phosphorylation upon ionizing radiation or UV radiation and identify Thr(714) as the in vivo BARD1 phosphorylation site. Importantly, DNA damage functions of BARD1 (i.e., inhibition of pre-mRNA polyadenylation and degradation of RNA polymerase II) are abrogated in T714A and T734A mutants. Our findings suggest that phosphorylation of BARD1 is critical for the DNA damage functions of the BRCA1/BARD1 complex.

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

The mechanisms involved in the p53-dependent control of gene expression following DNA damage have not been completely elucidated. Here, we show that the p53 C terminus associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3′ cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the 3′ processing factor cleavage-stimulation factor 1 (CstF1). We found that p53 can coexist in complexes with CstF and BARD1 in extracts of UV-treated cells, suggesting a role for p53 in mRNA 3′ cleavage following DNA damage. Consistent with this, we found that p53 inhibits 3′ cleavage in vitro and that there is a reverse correlation between the levels of p53 expression and the levels of mRNA 3′ cleavage under different cellular conditions. Supporting these results, a tumor-associated mutation in p53 not only decreases the interaction with BARD1 and CstF, but also decreases the UV-induced inhibition of 3′ processing, all of which is restored by wild-type-p53 expression. We also found that p53 expression levels affect the polyadenylation levels of housekeeping genes, but not of p21 and c-fos genes, which are involved in the DNA damage response (DDR). Here, we identify a novel 3′ RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network.

Multiple Properties of the Splicing Repressor SRp38 Distinguish It from Typical SR Proteins

ABSTRACT 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.

Positive and negative feedback loops in the p53 and mRNA 3′ processing pathways

Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. Recent studies have shown regulation of p53 at the mRNA level mediated via both the 5′ and the 3′ untranslated regions and affecting the stability and translation efficiency of the p53 mRNA. Here, we provide evidence of a feedback loop between p53 and the poly(A)-specific ribonuclease (PARN), in which PARN deadenylase keeps p53 levels low in nonstress conditions by destabilizing p53 mRNA, and the UV-induced increase in p53 activates PARN deadenylase, regulating gene expression during DNA damage response in a transactivation-independent manner. This model is innovative because it provides insights into p53 function and the mechanisms behind the regulation of mRNA 3′ end processing in 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. Copyright

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.

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 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ 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 5ʹ 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.