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Mutation or deletion of the U1 snRNP-associated factor LUC7L2 is associated with myeloid neoplasms, and knockout of LUC7L2 alters cellular metabolism. Here, we show that members of the LUC7 protein family differentially regulate two major classes of 5′ splice sites (5′SS) and broadly regulate mRNA splicing in both human cell lines and leukemias with LUC7L2 copy number variation. We describe distinctive 5′SS features of exons impacted by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5′SS with stronger consensus matching on the intron side of the near invariant /GU, while LUC7L3 enhances splicing of “left-handed” 5′SS with stronger consensus matching upstream of the /GU. We validated our model of sequence-specific 5′SS regulation both by mutating splice sites and swapping domains between human LUC7 proteins. Evolutionary analysis indicates that the LUC7L2/LUC7L3 subfamilies evolved before the split between animals and plants. Analysis of Arabidopsis thaliana mutants confirmed that plant LUC7 orthologs possess similar specificity to their human counterparts, indicating that 5′SS regulation by LUC7 proteins is highly conserved. Non-coding introns are excised from RNA transcripts of most eukaryotic genes by splicing. Here, Kenny et al. show that LUC7 proteins promote the splicing of different subsets of introns with distinct 5′ splice site sequences, and that these preferences are conserved from plants to human.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome 1 . Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs. A combination of five assays is used to produce a catalogue of RNA elements to which RNA-binding proteins bind in human cells.

Abstract Transposable elements (TEs) are self-replicating “genetic parasites” ubiquitous to eukaryotic genomes. In addition to conflict between TEs and their host genomes, TEs of the same family are in competition with each other. They compete for the same genomic niches while experiencing the same regime of copy-number selection. This suggests that competition among TEs may favor the emergence of new variants that can outcompete their ancestral forms. To investigate the sequence evolution of TEs, we developed a method to infer clades: collections of TEs that share SNP variants and represent distinct TE family lineages. We applied this method to a panel of 85 Drosophila melanogaster genomes and found that the genetic variation of several TE families shows significant population structure that arises from the population-specific expansions of single clades. We used population genetic theory to classify these clades into younger versus older clades and found that younger clades are associated with a greater abundance of sense and antisense piRNAs per copy than older ones. Further, we find that the abundance of younger, but not older clades, is positively correlated with antisense piRNA production, suggesting a general pattern where hosts preferentially produce antisense piRNAs from recently active TE variants. Together these findings suggest a pattern whereby new TE variants arise by mutation and then increase in copy number, followed by the host producing antisense piRNAs that may be used to silence these emerging variants.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03067-w

Eukaryotic genomes are replete with repeated sequences, in the form of transposable elements (TEs) dispersed across the genome or as satellite arrays, large stretches of tandemly repeated sequence. Many satellites clearly originated as TEs, but it is unclear how mobile genetic parasites can transform into megabase-sized tandem arrays. Comprehensive population genomic sampling is needed to determine the frequency and generative mechanisms of tandem TEs, at all stages from their initial formation to their subsequent expansion and maintenance as satellites. The best available population resources, short-read DNA sequences, are often considered to be of limited utility for analyzing repetitive DNA due to the challenge of mapping individual repeats to unique genomic locations. Here we develop a new pipeline called ConTExt which demonstrates that paired-end Illumina data can be successfully leveraged to identify a wide range of structural variation within repetitive sequence, including tandem elements. Analyzing 85 genomes from five populations of Drosophila melanogaster we discover that TEs commonly form tandem dimers. Our results further suggest that insertion site preference is the major mechanism by which dimers arise and that, consequently, dimers form rapidly during periods of active transposition. This abundance of TE dimers has the potential to provide source material for future expansion into satellite arrays, and we discover one such copy number expansion of the DNA transposon Hobo to ~16 tandem copies in a single line. The very process that defines TEs - transposition - thus regularly generates sequences from which new satellites can arise.

Typical RNAseq experiments uncover hundreds of splicing changes, reflecting underlying changes in splicing factor (SF) activity. Understanding transcriptomic variation in terms of SF activity requires elucidating the rules by which each SF impacts splicing. Here we present an interpretable regression model, KATMAP, which models splicing changes transcriptome-wide in terms of changes in SF binding and resulting altered regulation. The regulatory principles KATMAP learns generalize to predict the SF’s regulation at individual exons, with potential for design of splice-switching antisense oligonucleotides and inference of the displaced factor. We also discover cooperative splicing regulation by QKI and RFBOX proteins. KATMAP interprets RNAseq data by uncovering the factors responsible for transcriptomic changes, distinguishing direct SF targets from indirect effect, and infers relevant SFs from clinical RNAseq data.

Spliceosomal introns are a ubiquitous feature of eukaryotic genes, whose presence often boosts the expression of their host gene, a phenomenon known as intron-mediated enhancement (IME). IME has been noted across diverse genes and organisms, but remains mysterious in many respects. For example, how does intron sequence affect the magnitude of IME? In this study, we performed a massively parallel reporter assay (MPRA) to assess the effect of varying intron sequence on gene expression in a high-throughput manner, in human cells, using tens of thousands of synthetic introns with natural splice sites and randomized internal sequence. We observe that most random introns splice efficiently and enhance gene expression as well as or better than fully natural introns. Nearly all introns stimulate gene expression ∼eight-fold above an intronless control, at both mRNA and protein levels, suggesting that the primary mechanism acts to increase mRNA levels. IME strength is positively associated with splicing efficiency and with the intronic content of poly-uridine stretches, which we confirm using reporter experiments. Together, this work elucidates sequence determinants of IME from tens of thousands of random introns, and confirms that enhancement of gene expression is a general property of splicing.

Human LUC7 family proteins associate with the U1 small nuclear ribonucleoprotein (snRNP) complex. Mutation or deletion of LUC7L2 is associated with myeloid neoplasms, and depletion of LUC7L2 alters cellular metabolism. Here, we describe distinctive 5' splice site (5'SS) features of exons impacted by each of the three human LUC7s. We find that LUC7L2 and LUC7L enhance splicing of 'right-handed' 5'SS with stronger consensus matching on the intron side of the near-invariant /GU, while LUC7L3 preferentially enhances splicing of 'left-handed' 5'SS with stronger consensus matching on the exon side of the splice junction. Specificity for right- or left-handed 5'SS is conferred by the distinct structured N-terminal domains of LUC7L2 and LUC7L3. Evolutionary analysis shows that divergence of LUC7L3 and LUC7L2 subfamilies occurred prior to the divergence of plants from animals/fungi, and suggests that loss of the LUC7L3 subfamily from the fungal lineage contributed to the predominance of right-handed 5'SS in fungi.Competing Interest StatementThe authors have declared no competing interest.

Mutation or deletion of the U1 snRNP-associated factor LUC7L2 is associated with myeloid neoplasms, and knockout of LUC7L2 alters cellular metabolism. Here, we uncover that members of the LUC7 protein family differentially regulate two major classes of 5’ splice sites (5’SS) and broadly regulate mRNA splicing in both human cell lines and leukemias with LUC7L2 copy number variation. We describe distinctive 5’SS features of exons impacted by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5’SS with stronger consensus matching on the intron side of the near-invariant /GU, while LUC7L3 enhances splicing of “left-handed” 5’SS with stronger consensus matching upstream of the /GU. We validated our model of sequence-specific 5’SS regulation both by mutating splice sites and swapping domains between human LUC7 proteins. Evolutionary analysis indicates that the LUC7L2/LUC7L3 subfamilies diverged before the divergence of animals and plants. Analysis of Arabidopsis thaliana mutants confirmed that plant LUC7 orthologs possess specificity similar to their human counterparts, indicating that 5’SS regulation by LUC7 proteins is deeply conserved.

ABSTRACT Drosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85 D. melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we suggest instead that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than a third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes with niche specialization and telomere instability being previously underappreciated and likely predominant. Competing Interest Statement The authors have declared no competing interest. Footnotes * (mpm289{at}cornell.edu), (anne-marie.dion-cote{at}umoncton.ca), (barbash{at}cornell.edu) * This version has major revisions to the text and figures.