High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules.

While splicing changes caused by somatic mutations in SF3B1 are known, identifying fulllength isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

Activated PHO5 promoter chromatin at steady state represents a statistical ensemble of distinct structures.The extent of promoter nucleosome loss depends on the strength of the transcriptional activator of PHO5, indicative of continuous disassembly and reassembly of nucleosomes at the induced promoter.PHO5 promoter nucleosome loss and expression are exponentially related, pointing at two or more steps of the expression process that are activator controlled.The intrinsic noise profile of PHO5 expression permits quantitative distinction between alternative regulatory architectures.The assumption of two activator-controlled steps, promoter nucleosome removal and assembly of the transcription machinery, is necessary and sufficient to account for the quantitative relationship between PHO5 expression, intrinsic noise, and promoter nucleosome loss.

SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 have been identified with short-read RNA sequencing, a critical barrier in understanding the functional consequences of these splicing changes is that we lack the full transcript context in which these changes are occurring. Using nanopore sequencing technology, we have resequenced full-length cDNA from CLL samples with and without the hotspot SF3B1 K700E mutation, and a normal B cell. We have developed a workflow called FLAIR (Full-Length Alternative Isoform analysis of RNA), leveraging the full-length transcript sequencing data that nanopore affords. We report results from nanopore sequencing that are concordant with known SF3B1 biology from short read sequencing as well as altered intron retention events more confidently observed using long reads. Splicing analysis of nanopore reads between the SF3B1 WT and SF3B1 K700E samples identifies alternative upstream 3' splice sites associated with SF3B1 K700E . We also find downregulation of intron retention events in SF3B1 K700E relative to SF3B1 WT and no difference between CLL SF3B1 MT and B cell, suggesting an aberrant intron retention landscape in CLL samples lacking SF3B1 mutation. With full-length isoforms, we are able to better estimate the abundance of RNA transcripts that are productive and will likely be translated versus those that are unproductive. Validation from short-read data also reveals a strong branch point sequence in these downregulated intron retention events, consistent with previously reported branch points associated with mutated SF3B1. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

During the estrous cycle and beginning in estrus, the mammary gland undergoes pregnancy-like development that depends on transcriptional regulation by the estrogen and progesterone receptors (ER, PR) and Pax-2 as well as the action of the growth factors Wnt-4 and RANKL. In this report, we first describe the decay and delayed expression of ERa, PR, and Pax-2 proteins as well as depression of Wnt-4 and RANKL mRNA coincident with the strong estrogen surge in proestrus. In time-course studies using ovariectomized mice, a single estrogen injection replicated these delays and caused an 18 h delay in Wnt-4 expression. Molecular time-delay systems are at the core of cellular cycles, most notably the circadian clock, and depend on proteasome degradation of transcriptional regulators that exhibit dedicated timing functions. The cytoplasmic dynamics of these regulators govern delay duration through negative transcription/translation feedback loops. A proteasome inhibitor, PS-341, blocked estrogen-stimulated ERa, PR, and Pax-2 decay and proteasome chymotryptic activity, assayed using a fluorogenic substrate, was elevated in proestrus correlating with the depletion of the transcription factors. The 18-h delay in Wnt-4 induction corresponded to the turnover time of Pax-2 protein in the cytoplasm and was eliminated in Pax-2 knockout mammary tissue, demonstrating that Pax-2 has a unique timing function. The patterns of estrogen-triggered ERa, PR, and Pax-2 turnover were consistent with a negative transcriptional feedback. Retarding the expression of ERa, PR, and Pax-2 may optimize preparations for pregnancy by coordinating expression of critical receptors and transcription factors with rising estrogen and progesterone levels in estrus. The estrogen surge in proestrus has no defined mammotropic function. This study provides the first evidence that it is a synchronizing signal triggering proteasome-dependent turnover of mammary gland ERa, PR, and Pax-2. We hypothesize that the delays reflect a previously unrecognized timing system, which is present in all ovarian target tissues.