Effect of mismatch repair on the mutational footprint of the bacterial SOS mutator activity.

The bacterial SOS response to DNA damage induces an error-prone repair program that is mutagenic. In Escherichia coli, SOS-induced mutations are caused by the translesion synthesis (TLS) activity of two error-prone polymerases (EPPs), Pol IV and Pol V. The mutational footprint of the EPPs is confounded by both DNA damage and repair, as mutations are targeted to DNA lesions via TLS and corrected by the mismatch repair (MMR) system. To remove these factors and assess untargeted EPP mutations genome-wide, we constructed spontaneous SOS mutator strains deficient in MMR, then analyzed their mutational footprints by mutation accumulation and whole genome sequencing. Our analysis reveals new features of untargeted SOS-mutagenesis, showing how MMR alters its spectrum, sequence specificity, and strand-bias. Our data support a model where the EPPs prefer to act on the lagging strand of the replication fork, producing base pair mismatches that are differentially repaired by MMR depending on the type of mismatch.

Subcellular RNA sequencing reveals broad presence of cytoplasmic intron-sequence retaining transcripts in mouse and rat neurons.

Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-ß mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways.