A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications.

Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs as well as the spliceosomal protein Prp8 in eukaryotes. Here we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming deoxynucleotide triphosphate (dNTP) at 3.0-Å resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs but remarkably similar to viral RNA-dependent RNA polymerases. The structure reveals a host of features not seen previously in RTs that may contribute to distinctive biochemical properties of group II intron RTs, and it provides a prototype for many related bacterial and eukaryotic non-LTR retroelement RTs. It also reveals how protein structural features used for reverse transcription evolved to promote the splicing of both group II and spliceosomal introns.

Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase.

Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template-DNA primer heteroduplex with a 1-nt 3'-DNA overhang. The structure showed that the 3' end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non-long terminal repeat-retroelement RTs and the RT fingertips loop, with the 3' nucleotide of the acceptor base paired to the 1-nt 3'-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non-long terminal repeat-retroelement RTs and viral RNA-dependent RNA polymerases.

A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing.

The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5'-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5'-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5'-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.

Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6.

Glycogen synthase kinase-3 (GSK-3) is a key regulator of many cellular signaling pathways. Unlike most kinases, GSK-3 is controlled by inhibition rather than by specific activation. In the insulin and several other signaling pathways, phosphorylation of a serine present in a conserved sequence near the amino terminus of GSK-3 generates an auto-inhibitory peptide. In contrast, Wnt/ß-catenin signal transduction requires phosphorylation of Ser/Pro rich sequences present in the Wnt co-receptors LRP5/6, and these motifs inhibit GSK-3 activity. We present crystal structures of GSK-3 bound to its phosphorylated N-terminus and to two of the phosphorylated LRP6 motifs. A conserved loop unique to GSK-3 undergoes a dramatic conformational change that clamps the bound pseudo-substrate peptides, and reveals the mechanism of primed substrate recognition. The structures rationalize target sequence preferences and suggest avenues for the design of inhibitors selective for a subset of pathways regulated by GSK-3. DOI: http://dx.doi.org/10.7554/eLife.01998.001.

The ß-catenin destruction complex.

The Wnt/ß-catenin pathway is highly regulated to insure the correct temporal and spatial activation of its target genes. In the absence of a Wnt stimulus, the transcriptional coactivator ß-catenin is degraded by a multiprotein "destruction complex" that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase ß-TrCP. The complex generates a ß-TrCP recognition site by phosphorylation of a conserved Ser/Thr-rich sequence near the ß-catenin amino terminus, a process that requires scaffolding of the kinases and ß-catenin by Axin. Ubiquitinated ß-catenin is degraded by the proteasome. The molecular mechanisms that underlie several aspects of destruction complex function are poorly understood, particularly the role of APC. Here we review the molecular mechanisms of destruction complex function and discuss several potential roles of APC in ß-catenin destruction.

Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/beta-catenin signaling.

Wnt/beta-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3beta bound to the scaffold protein Axin. However, it remains unknown how GSK3beta is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3beta in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3beta in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3beta. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3beta using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.

Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation.

The transcriptional coactivator beta-catenin mediates Wnt growth factor signaling. In the absence of a Wnt signal, casein kinase 1 (CK1) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate cytosolic beta-catenin, thereby flagging it for recognition and destruction by the ubiquitin/proteosome machinery. Phosphorylation occurs in a multiprotein complex that includes the kinases, beta-catenin, axin, and the Adenomatous Polyposis Coli (APC) protein. The role of APC in this process is poorly understood. CK1epsilon and GSK-3beta phosphorylate APC, which increases its affinity for beta-catenin. Crystal structures of phosphorylated and nonphosphorylated APC bound to beta-catenin reveal a phosphorylation-dependent binding motif generated by mutual priming of CK1 and GSK-3beta substrate sequences. Axin is shown to act as a scaffold for substrate phosphorylation by these kinases. Phosphorylated APC and axin bind to the same surface of, and compete directly for, beta-catenin. The structural and biochemical data suggest a novel model for how APC functions in beta-catenin degradation.