A Link between Intronic Polyadenylation and HR Maintenance Discovered

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Homologous recombination (HR) is one of the most complicated DNA repair pathways in eukaryotic cells. More than a hundred genes and their expression products are involved in the HR process. The BRCAness mutations are a typical family of HR mutations that are responsible for the collapse of replication forks. (1) Among the BRCAness genes, CDK12 is the only nondirectly functional gene in the HR pathway. As a kinase, CDK12 phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) and has an apparent regulatory role in the transcription and maturation of certain genes, but how CDK12 regulates HR genes was totally unknown until Dubbury et al. revealed its function to suppress in intronic polyadenylation. (2)

Unlike the genes that function directly in the HR pathway, CDK12 cannot be studied by conventional cell biology or biochemistry approaches with clear phenotypes in the HR process. Dubbury et al. designed an elegant model system using the mouse embryonic stem (mES) cells to mimic the defective HR repair induced by the withdrawal of doxycycline to deplete CDK12 proteins from the cells. These allowed the investigation of molecular consequences upon CDK12 loss in a systematic level.

Using RNA sequencing, Dubbury et al. analyzed the transcript profiles of the model mES cell CDK12 depletion. Most of the genes with significant changes in gene expressions were secondary effects of CDK12 depletion on either p53 activation or bivalent chromatin modifications. Only 3% of expressed genes were modestly affected with a slight decrease in their gene expression levels upon CDK12 loss. This indicated that CDK12’s regulatory function on HR is not through the change of the gene expression level. There are other causes that may affect the variation of functional gene products such as alternative splicing or alternative polyadenylation, which are also regulatory hallmarks of the phosphorylation of RNAPII by CDK12. (3,4) Further analysis of the RNA transcripts revealed that the depletion of CDK12 had a very little effect on splicing but a strong influence on alternative polyadenylation.

Two distinct types of polyadenylation of mRNAs are, respectively, the distal polyadenylation after the 3′-most exon of a gene and the intronic polyadenylation (IPA) in the introns of a gene. Using the 3′-end sequencing data, Dubbury et al. found that CDK12-depleted mES cells demonstrated a global increase of the IPA sites and decrease of the distal polyadenylation sites. Quantitative analysis indicated that the decrease of distal polyadenylation was caused by the upstream IPA in the corresponding genes. Furthermore, more IPA isoform usage appeared to cause less distal polyadenylation isoforms. These results implicated that CDK12 is responsible for suppressing IPA sites genome-wide, therefore promoting more full-length mRNA products.

How does the phosphorylation of the RNAPII by CDK12 affect the alternative polyadenylation? Because the alternative polyadenylation occurs co-transcriptionally, the transcriptional status and interaction of RNAPII on its transcribing genes may influence the cleavage and polyadenylation machinery on the RNA transcripts. Using chromatin immunoprecipitation sequencing (ChIP) and a novel in-house bioinformatics and statistical method for metagene profiling, Dubbury et al. investigated the unphosphorylated RNAPII and Ser2p RNAPII distribution on the genomes of mES cells with and without CDK12 depletion, respectively. They indeed discovered a density change of RNAPII from the transcriptional start site to the distal polyadenylation site of genes in a genome-wide scale upon the loss of CDK12. Specifically, the density of RNAPII decreased at the 5′ ends of genes but steadily increased toward the 3′ ends. A more precise analysis revealed that, upon CDK12 loss, the RNAPII density increased upstream and decreased downstream of the first nucleosome from the 5′ ends of genes. This indicated a reduction of RNAPII in the transcription elongation status in the cell’s lack of CDK12. Such an RNAPII density change is not only specific to the genes with a significant alternative polyadenylation but also observed in other genes. Therefore, the CDK12 phosphorylation on the CTD of RNAPII affects the transcription elongation status genome-wide. It has been known that phosphorylation of RNAPII’s CTD is involved in the regulation of splicing and alternative polyadenylation activities. (5) CDK12’s phosphorylation of serine 2 (Ser2p) of RNAPII’s CTD heptapeptide repeat could directly inhibit the IPA site usage. The phosphorylation may also promote the splicing activity, which competes off of the IPA site usage. The Ser2p of RNAPII’s CTD also promotes the elongation activity, potentially resulting in a shift of the kinetic balance toward less usage of the IPA sites. All or a combination of the above possibilities may contribute to CDK12’s suppression of the IPA usage during transcription.

Are the HR genes among those genes with enhanced IPA usage upon CDK12 loss? The experimental data by Dubbury et al. demonstrated statistically significant increases of IPA usage or decreases in distal polyadenylation usage for the BRCAness group of genes in the cells depleted of CDK12. A closer examination revealed that these CDK12-sensitive HR genes indeed harbor more IPAs. Furthermore, for an unknown reason, these HR genes are more sensitive to CDK12 loss than other expressed non-HR genes with the same number of IPAs. The strong IPA usage of the HR genes accounted for a substantial reduction of the corresponding full-length proteins in the CDK12-depleted cells. CDK12, therefore, plays an important role in maintaining the expression of many BRCAness genes to generate full-length matured proteins (Figure 1). This function of CDK12’s phosphorylation was underscored by further validation from the CDK12 loss-of-function point mutations and deletions in various human cancerous tumor cells. Therefore, in a conserved mechanism, CDK12 suppresses intronic polyadenylation of critical HR genes by phosphorylating the serine 2 of RNAPII’s CTD.

Figure 1

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The link between the intronic polyadenylation and the HR maintenance discovered by the bioinformatics approach is just a beginning of this fascinating pathway. A lot of questions remain to be answered, such as the molecules involved in this link, the detail mechanism of the suppression by CKD12 on IPA, the reasons of BRCAness genes’ high sensitivity on CDK12 depletion and structural basis of these processes. More in-depth studies using multiple approaches such as biochemistry, biophysics and structural biology in the future will shed light on these questions.