Crystal structure and functional properties of the human CCR4-CAF1 deadenylase complex.

The CCR4 and CAF1 deadenylases physically interact to form the CCR4-CAF1 complex and function as the catalytic core of the larger CCR4-NOT complex. Together, they are responsible for the eventual removal of the 3'-poly(A) tail from essentially all cellular mRNAs and consequently play a central role in the posttranscriptional regulation of gene expression. The individual properties of CCR4 and CAF1, however, and their respective contributions in different organisms and cellular environments are incompletely understood. Here, we determined the crystal structure of a human CCR4-CAF1 complex and characterized its enzymatic and substrate recognition properties. The structure reveals specific molecular details affecting RNA binding and hydrolysis, and confirms the CCR4 nuclease domain to be tethered flexibly with a considerable distance between both enzyme active sites. CCR4 and CAF1 sense nucleotide identity on both sides of the 3'-terminal phosphate, efficiently differentiating between single and consecutive non-A residues. In comparison to CCR4, CAF1 emerges as a surprisingly tunable enzyme, highly sensitive to pH, magnesium and zinc ions, and possibly allowing distinct reaction geometries. Our results support a picture of CAF1 as a primordial deadenylase, which gets assisted by CCR4 for better efficiency and by the assembled NOT proteins for selective mRNA targeting and regulation.

Human LINE-1 retrotransposition requires a metastable coiled coil and a positively charged N-terminus in L1ORF1p.

LINE-1 (L1) is an autonomous retrotransposon, which acted throughout mammalian evolution and keeps contributing to human genotypic diversity, genetic disease and cancer. L1 encodes two essential proteins: L1ORF1p, a unique RNA-binding protein, and L1ORF2p, an endonuclease and reverse transcriptase. L1ORF1p contains an essential, but rapidly evolving N-terminal portion, homo-trimerizes via a coiled coil and packages L1RNA into large assemblies. Here, we determined crystal structures of the entire coiled coil domain of human L1ORF1p. We show that retrotransposition requires a non-ideal and metastable coiled coil structure, and a strongly basic L1ORF1p amino terminus. Human L1ORF1p therefore emerges as a highly calibrated molecular machine, sensitive to mutation but functional in different hosts. Our analysis rationalizes the locally rapid L1ORF1p sequence evolution and reveals striking mechanistic parallels to coiled coil-containing membrane fusion proteins. It also suggests how trimeric L1ORF1p could form larger meshworks and indicates critical novel steps in L1 retrotransposition.

Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells.

Mobilization of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution, but these mutagenic events can also cause genetic disorders. In humans, retrotransposon mobilization is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilize in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilization. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, loss of Tex19.1 increases L1-ORF1p levels and L1 mobilization in pluripotent mouse embryonic stem cells, implying that Tex19.1 prevents de novo retrotransposition in the pluripotent phase of the germline cycle. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in mammals.

Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition.

Non-LTR retrotransposons are mobile genetic elements and play a major role in eukaryotic genome evolution and disease. Similar to retroviruses they encode a reverse transcriptase, but their genomic integration mechanism is fundamentally different, and they lack homologs of the retroviral nucleocapsid-forming protein Gag. Instead, their first open reading frames encode distinct multi-domain proteins (ORF1ps) presumed to package the retrotransposon-encoded RNA into ribonucleoprotein particles (RNPs). The mechanistic roles of ORF1ps are poorly understood, particularly of ORF1ps that appear to harbor an enzymatic function in the form of an SGNH-type lipolytic acetylesterase. We determined the crystal structures of the coiled coil and esterase domains of the ORF1p from the Danio rerio ZfL2-1 element. We demonstrate a dimerization of the coiled coil and a hydrolytic activity of the esterase. Furthermore, the esterase binds negatively charged phospholipids and liposomes, but not oligo-(A) RNA. Unexpectedly, the esterase can split into two dynamic half-domains, suited to engulf long fatty acid substrates extending from the active site. These properties indicate a role for lipids and membranes in non-LTR retrotransposition. We speculate that Gag-like membrane targeting properties of ORF1ps could play a role in RNP assembly and in membrane-dependent transport or localization processes.

Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition.

The LINE-1 (L1) retrotransposon emerges as a major source of human interindividual genetic variation, with important implications for evolution and disease. L1 retrotransposition is poorly understood at the molecular level, and the mechanistic details and evolutionary origin of the L1-encoded L1ORF1 protein (L1ORF1p) are particularly obscure. Here three crystal structures of trimeric L1ORF1p and NMR solution structures of individual domains reveal a sophisticated and highly structured, yet remarkably flexible, RNA-packaging protein. It trimerizes via an N-terminal, ion-containing coiled coil that serves as scaffold for the flexible attachment of the central RRM and the C-terminal CTD domains. The structures explain the specificity for single-stranded RNA substrates, and a mutational analysis indicates that the precise control of domain flexibility is critical for retrotransposition. Although the evolutionary origin of L1ORF1p remains unclear, our data reveal previously undetected structural and functional parallels to viral proteins.

Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame.

Non-LTR retrotransposons (NLRs) are a unique class of mobile genetic elements that have significant impact on the evolution of eukaryotic genomes. However, the molecular details and functions of their encoded proteins, in particular of the accessory ORF1p proteins, are poorly understood. Here, we identify noncanonical RNA-recognition-motifs (RRMs) in several phylogenetically unrelated NLR ORF1p proteins. This provides an explanation for their RNA-binding properties and clearly shows that they are not related to the retroviral nucleocapsid protein Gag, despite the frequent presence of CCHC zinc knuckles. In particular, we characterize the ORF1p protein of the human long interspersed nuclear element 1 (LINE-1 or L1). We show that L1ORF1p is a multidomain protein, consisting of a coiled coil (cc), RRM, and C-terminal domain (CTD). Most importantly, we solved the crystal structure of the RRM domain, which is characterized by extended loops stabilized by unique salt bridges. Furthermore, we demonstrate that L1ORF1p trimerizes via its N-terminal cc domain, and we suggest that this property is functionally important for all homologues. The formation of distinct complexes with single-stranded nucleic acids requires the presence of the RRM and CTD domains on the same polypeptide chain as well as their close cooperation. Finally, the phylogenetic analysis of mammalian L1ORF1p shows an ancient origin of the RRM domain and supports a modular evolution of NLRs.