Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators.

Transposable elements, also known as transposons, are now recognized not only as parasitic DNA, the spread of which in the genome must be controlled by the host, but also as major players in genome evolution and regulation. Long interspersed element-1 (LINE-1, also known as L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and generates inter- and intra-individual genetic variation, in some cases resulting in disease. However, how L1 activity is controlled and the function of L1s in host gene regulation are not completely understood. Here we use CRISPR-Cas9 screening strategies in two distinct human cell lines to provide a genome-wide survey of genes involved in the control of L1 retrotransposition. We identify functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, which are often associated with human diseases, control the L1 life cycle at the transcriptional or the post-transcriptional level in a manner that can depend on the endogenous L1 nucleotide sequence, underscoring the complexity of L1 regulation. We further investigate the restriction of L1 by the protein MORC2 and by the human silencing hub (HUSH) complex subunits MPP8 and TASOR. HUSH and MORC2 can selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environments, and promote deposition of histone H3 Lys9 trimethylation (H3K9me3) for transcriptional silencing. Notably, these silencing events often occur within introns of transcriptionally active genes, and lead to the downregulation of host gene expression in a HUSH-, MORC2-, and L1-dependent manner. Together, these results provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway and illustrate how epigenetic silencing of transposable elements rewires host gene expression programs.

Translation readthrough mitigation.

A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR­Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3' untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3' UTR sequences to reduce mature protein expression in tissue culture assays, including 3' UTR sequences from the hypomorphic 'Constant Spring' haemoglobin stop codon variant. We suggest that 3' UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

The integral spliceosomal component CWC15 is required for development in Arabidopsis.

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.