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.

A shape-shifting nuclease unravels structured RNA.

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3'-5' exoribonuclease that plays a critical role in human development. Dis3L2 independently degrades structured substrates, including coding and noncoding 3' uridylated RNAs. While the basis for Dis3L2's substrate recognition has been well characterized, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining cryogenic electron microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for RNA processing. We discovered a dramatic conformational change that is triggered by double-stranded RNA (dsRNA), repositioning two cold shock domains by 70 Å. This movement exposes a trihelix linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not single-stranded RNA, degradation. These findings reveal the conformational plasticity of Dis3L2 and detail a mechanism of structured RNA degradation.

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.

Poly(A) tail dynamics: Measuring polyadenylation, deadenylation and poly(A) tail length.

Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is under constant regulation by cap-binding proteins, miRNAs, and RNA binding proteins, including poly(A) binding proteins (PABPs). The interplay between all these factors determines whether nuclear or cytoplasmic exoribonucleases will gain access to and remove the poly(A) tail, which is so critical to the stability and translation capacity of the mRNA. In this chapter, we present an overview of two of the key features of the mRNA life-cycle: cleavage/polyadenylation and deadenylation, and describe biochemical assays that have been generated to study the activity of each of these enzymatic reactions. Finally, we also provide protocols to investigate mRNA's poly(A) length. The importance of these assays is highlighted by the dynamic and essential role the poly(A) tail length plays in controlling gene expression.