In spliceosome assembly, the 5 splice site is initially recognized by U1 snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to the maintenance of 5 splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in Caenorhabditis elegans at SNRP-27 M141 promote changes in alternative 5ss usage. We tested whether the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining 5 splice site identity during spliceosome assembly. We find that SNU-66 H765 mutants promote alternative 5 splice site usage. Many of the alternative 5 splicing events affected by SNU-66(H765G) overlap with those affected SNRP-27(M141T). Double mutants of snrp-27(M141T) and snu-66(H765G) are homozygous lethal. We hypothesize that mutations at either SNRP-27 M141 or SNU-66 H765 allow the spliceosome to load alternative 5 splice sites into the active site. Tests with mutant U1 snRNA and swapped 5 splice sites indicate that the ability of SNRP-27 M141 and SNU-66 H765 mutants to affect a particular 5 splice alternative splicing event is dependent on both the presence of a weaker consensus 5ss nearby and potentially nearby splicing factor binding sites. Our findings confirm a new role for the C terminus of SNU-66 in maintenance of 5 splice site identity during spliceosome assembly.

During development of the cerebral cortex, neural stem cells divide to expand the progenitor pool and generate basal progenitors, outer radial glia, and cortical neurons. As these newly born neurons differentiate, they must properly migrate toward their final destination in the cortical plate, project axons to appropriate targets, and develop dendrites. However, a complete understanding of the precise genetic mechanisms regulating these steps is lacking. Here we show that a member of the nuclear factor one (NFI) family of transcription factors, NFIB, is essential for many of these processes in mice. We performed a detailed analysis of NFIB expression during cortical development, and investigated defects in cortical neurogenesis, neuronal migration, and differentiation in NfiB(-/-) brains. We found that NFIB is strongly expressed in radial glia and corticofugal neurons throughout cortical development. However, in NfiB(-/-) cortices, radial glia failed to generate outer radial glia, subsequently resulting in a loss of late basal progenitors. In addition, corticofugal neurons showed a severe loss of axonal projections, whereas late-born cortical neurons displayed defects in migration and ectopically expressed the early-born neuronal marker CTIP2. Furthermore, gene expression analysis, by RNA sequencing, revealed a misexpression of genes that regulate the cell cycle, neuronal differentiation and migration in NfiB(-/-) brains. Together these results demonstrate the critical functions of NFIB in regulating cortical development.

During its sexual reproduction, the stichotrichous ciliate Oxytricha trifallax orchestrates a remarkable transformation of one of the newly formed germline micronuclear genomes. Hundreds of thousands of gene pieces are stitched together, excised from chromosomes, and replicated dozens of times to yield a functional somatic macronuclear genome composed of ~16,000 distinct DNA molecules that typically encode a single gene. Little is known about the proteins that carry out this process. We profiled mRNA expression as a function of macronuclear development and identified hundreds of mRNAs preferentially expressed at specific times during the program. We find that a disproportionate number of these mRNAs encode proteins that are involved in DNA and RNA functions. Many mRNAs preferentially expressed during macronuclear development have paralogs that are either expressed constitutively or are expressed at different times during macronuclear development, including many components of the RNA polymerase II machinery and homologous recombination complexes. Hundreds of macronuclear development-specific genes encode proteins that are well-conserved among multicellular eukaryotes, including many with links to germline functions or development. Our work implicates dozens of DNA and RNA-binding proteins with diverse evolutionary trajectories in macronuclear development in O. trifallax. It suggests functional connections between the process of macronuclear development in unicellular ciliates and germline specialization and differentiation in multicellular organisms, and argues that gene duplication is a key source of evolutionary innovation in this process.

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3 splice site (3ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3 splicing in introns with pairs of 3 splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3 splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3 splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3ss.

Fast evolving regions of many metazoan genomes show a bias toward substitutions that change weak (A,T) into strong (G,C) base pairs. Single-nucleotide polymorphisms (SNPs) do not share this pattern, suggesting that it results from biased fixation rather than biased mutation. Supporting this hypothesis, analyses of polymorphism in specific regions of the human genome have identified a positive correlation between weak to strong (W→S) SNPs and derived allele frequency (DAF), suggesting that SNPs become increasingly GC biased over time, especially in regions of high recombination. Using polymorphism data generated by the 1000 Genomes Project from 179 individuals from 4 human populations, we evaluated the extent and distribution of ongoing GC-biased evolution in the human genome. We quantified GC fixation bias by comparing the DAFs of W→S mutations and S→W mutations using a Mann-Whitney U test. Genome-wide, W→S SNPs have significantly higher DAFs than S→W SNPs. This pattern is widespread across the human genome but varies in magnitude along the chromosomes. We found extreme GC-biased evolution in neighborhoods of recombination hot spots, a significant correlation between GC bias and recombination rate, and an inverse correlation between GC bias and chromosome arm length. These findings demonstrate the presence of ongoing fixation bias favoring G and C alleles throughout the human genome and suggest that the bias is caused by a recombination-associated process, such as GC-biased gene conversion.

Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae, documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3 processing machinery) attacks the branch, joining the intron 3 end to the 5 splice site in a 3-5 linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

Generation of distinct cortical projection neuron subtypes during development relies in part on repression of alternative neuron identities. It was reported that the special AT-rich sequence-binding protein 2 (Satb2) is required for proper development of callosal neuron identity and represses expression of genes that are essential for subcerebral axon development. Surprisingly, Satb2 has recently been shown to be necessary for subcerebral axon development. Here, we unravel a previously unidentified mechanism underlying this paradox. We show that SATB2 directly activates transcription of forebrain embryonic zinc finger 2 (Fezf2) and SRY-box 5 (Sox5), genes essential for subcerebral neuron development. We find that the mutual regulation between Satb2 and Fezf2 enables Satb2 to promote subcerebral neuron identity in layer 5 neurons, and to repress subcerebral characters in callosal neurons. Thus, Satb2 promotes the development of callosal and subcerebral neurons in a cell context-dependent manner.

During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing.

Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28-29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2'O-methylation at their 3' ends. We propose models for the role of these "27macRNAs" in macronuclear development.

The tri-snRNP 27K protein is a component of the human U4/U6-U5 tri-snRNP and contains an N-terminal phosphorylated RS domain. In a forward genetic screen in C. elegans, we previously identified a dominant mutation, M141T, in the highly-conserved C-terminal region of this protein. The mutant allele promotes changes in cryptic 5 splice site choice. To better understand the function of this poorly characterized splicing factor, we performed high-throughput mRNA sequencing analysis on worms containing this dominant mutation. Comparison of alternative splice site usage between the mutant and wild-type strains led to the identification of 26 native genes whose splicing changes in the presence of the snrp-27 mutation. The changes in splicing are specific to alternative 5 splice sites. Analysis of new alleles suggests that snrp-27 is an essential gene for worm viability. We performed a novel directed-mutation experiment in which we used the CRISPR-cas9 system to randomly generate mutations specifically at M141 of SNRP-27. We identified eight amino acid substitutions at this position that are viable, and three that are homozygous lethal. All viable substitutions at M141 led to varying degrees of changes in alternative 5 splicing of native targets. We hypothesize a role for this SR-related factor in maintaining the position of the 5 splice site as U1snRNA trades interactions at the 5 end of the intron with U6snRNA and PRP8 as the catalytic site is assembled.