Quantitative immunopeptidomics reveals a tumor stroma-specific target for T cell therapy.

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

Regulated dicing of pre-mir-144 via reshaping of its terminal loop.

Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs.

Library Selection with a Randomized Repertoire of (ßα)8-Barrel Enzymes Results in Unexpected Induction of Gene Expression.

The potential of the frequently encountered (ßα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (ßα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same mutants of the E. coli genes nanE (encoding a putative N-acetylmannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5'-phosphate synthase) were responsible for rescuing both ΔserB and ΔserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactions in vitro. Instead, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of ΔserB and ΔserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (ßα)8-barrel fold.

The nuclear matrix protein Matr3 regulates processing of the synaptic microRNA-138-5p.

microRNA-dependent post-transcriptional control represents an important gene-regulatory layer in post-mitotic neuronal development and synaptic plasticity. We recently identified the brain-enriched miR-138 as a negative regulator of dendritic spine morphogenesis in rat hippocampal neurons. A potential involvement of miR-138 in cognition is further supported by a recent GWAS study on memory performance in a cohort of aged (>60 years) individuals. The expression of miR-138, which is encoded in two independent genomic loci (miR-138-1 and -2), is subject to both cell-type and developmental stage-specific regulation, the underlying molecular mechanisms however are poorly understood. Here, we show that miR-138-2 is the primary source of mature miR-138 in developing rat hippocampal neurons. Furthermore, we obtained evidence for the regulation of miR-138-2 biogenesis at the level of primary miRNA processing. Using biochemical pull-down assays, we identified the nuclear matrix protein Matrin-3 as pri/pre-miR-138 interacting protein and mapped the interaction to the pri/pre-miR-138-2 loop region. Matrin-3 loss-of-function experiments in HEK293 cells and primary neurons together with protein localization studies suggest an inhibitory function of Matrin-3 in nuclear pri-miR-138-2 processing. Together, our experiments unravel a new mechanism of miR-138 regulation in neurons, with important implications for miR-138 regulation during neuronal development, synaptic plasticity and memory-related processes.

Publisher Correction: Regulation of microRNA biogenesis and its crosstalk with other cellular pathways.

The legend of Figure 2 neglected to acknowledge that part b was adapted with permission from ref.46, Elsevier and that part d, third panel from the left was reproduced from ref.62, Springer Nature Limited. The change has been made in the HTML and PDF versions of the manuscript.

Regulation of microRNA biogenesis and its crosstalk with other cellular pathways.

MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. miRNAs are transcribed as precursor molecules, which are subsequently cleaved by the endoribonucleases Drosha and Dicer. Mature miRNAs are bound by a member of the Argonaute (AGO) protein family to form the RNA-induced silencing complex (RISC) in a process termed RISC loading. Advances in structural analyses of Drosha and Dicer complexes enabled elucidation of the mechanisms that drive these molecular machines. Transcription of miRNAs, their processing by Drosha and Dicer and RISC loading are key steps in miRNA biogenesis, and various additional factors facilitate, support or inhibit these processes. Recent work has revealed that regulatory factors not only coordinate individual miRNA processing steps but also connect miRNA biogenesis with other cellular processes. Protein phosphorylation, for example, links miRNA biogenesis to various signalling pathways, and such modifications are often associated with disease. Furthermore, not all miRNAs follow canonical processing routes, and many non-canonical miRNA biogenesis pathways have recently been characterized.

Author Correction: Regulation of microRNA biogenesis and its crosstalk with other cellular pathways.

In Figure 1b, the GHG sequence motif in the primary microRNA has been moved to the basal stem and the ruler of the basal stem has been shortened to more precisely delineate 11 base pairs. The changes have been made in the HTML and PDF versions of the manuscript.

Reconstitution of mammalian cleavage factor II involved in 3' processing of mRNA precursors.

Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

LIN28 Selectively Modulates a Subclass of Let-7 MicroRNAs.

LIN28 is a bipartite RNA-binding protein that post-transcriptionally inhibits the biogenesis of let-7 microRNAs to regulate development and influence disease states. However, the mechanisms of let-7 suppression remain poorly understood because LIN28 recognition depends on coordinated targeting by both the zinc knuckle domain (ZKD), which binds a GGAG-like element in the precursor, and the cold shock domain (CSD), whose binding sites have not been systematically characterized. By leveraging single-nucleotide-resolution mapping of LIN28 binding sites in vivo, we determined that the CSD recognizes a (U)GAU motif. This motif partitions the let-7 microRNAs into two subclasses, precursors with both CSD and ZKD binding sites (CSD+) and precursors with ZKD but no CSD binding sites (CSD-). LIN28 in vivo recognition-and subsequent 3' uridylation and degradation-of CSD+ precursors is more efficient, leading to their stronger suppression in LIN28-activated cells and cancers. Thus, CSD binding sites amplify the regulatory effects of LIN28.

Identification of microRNA Precursor-Associated Proteins.

MicroRNA (miRNA) biogenesis is regulated intricately at multiple levels. In addition to transcriptional control of pri-miRNA loci, sequence as well as structural features of the pri-miRNA-stem loop determine its processing efficiency by the endonucleases Drosha and Dicer. On the one hand, general features are necessary to allow a hairpin to be recognized by the processing machinery; on the other hand, specific sequence motifs of individual miRNA precursors can be read by RNA binding proteins (RBPs) that regulate processing, leading to increased or decreased levels of functional miRNAs. In a pulldown experiment using the pri-miRNA hairpin as immobilized bait, cognate RBPs can be isolated and analyzed by immunoblotting or mass spectrometry, allowing for the discovery or analysis of protein regulators of miRNA biogenesis.

Interactions, localization, and phosphorylation of the m6A generating METTL3-METTL14-WTAP complex.

N6-methyladenine (m6A) is found on many eukaryotic RNAs including mRNAs. m6A modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates m6A on mRNAs. METTL3/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. Although recent crystal structures revealed how the catalytic MT-A70 domains of METTL3 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. Here, we used recombinant proteins and mapped binding surfaces within the METTL3/14-WTAP complex. Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Using an in vitro methylation assay, we confirm that monomeric METTL3 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METTL3/14 activity by contributing to RNA substrate binding. Our biochemical work identifies characteristic features of METTL3/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex.

A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis.

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.

Structural and functional insights into the fly microRNA biogenesis factor Loquacious.

In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.

The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation.

TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.

Structure of an Ebf1:DNA complex reveals unusual DNA recognition and structural homology with Rel proteins.

Early B-cell factor 1 (Ebf1) is a key transcriptional determinant of B-lymphocyte differentiation whose DNA-binding domain has no sequence similarity to other transcription factor families. Here we report the crystal structure of an Ebf1 dimer bound to its palindromic recognition site. The DNA-binding domain adopts a pseudoimmunoglobulin-like fold with novel topology, but is structurally similar to the Rel homology domains of NFAT and NF-κB. Ebf1 contacts the DNA with two loop-based modules and a unique Zn coordination motif whereby each Ebf1 monomer interacts with both palindromic half-sites. This unusual mode of DNA recognition generates an extended contact area that may be crucial for the function of Ebf1 in chromatin.

SMADs stimulate miRNA processing.

MicroRNAs (miRNAs) are potent regulators of gene expression. Consequently, miRNA expression is tightly regulated itself. In this issue, Hata and coworkers provide mechanistic insights into how SMAD proteins regulated the biogenesis of a specific subset of human miRNAs.

Early B cell factor 1 regulates B cell gene networks by activation, repression, and transcription- independent poising of chromatin.

The transcription factor early B cell factor-1 (Ebf1) is a key determinant of B lineage specification and differentiation. To gain insight into the molecular basis of Ebf1 function in early-stage B cells, we combined a genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses. Among 565 genes that are occupied and transcriptionally regulated by Ebf1, we identified large sets involved in (pre)-B cell receptor and Akt signaling, cell adhesion, and migration. Interestingly, a third of previously described Pax5 targets was found to be occupied by Ebf1. In addition to Ebf1-activated and -repressed genes, we identified targets at which Ebf1 induces chromatin changes that poise the genes for expression at subsequent stages of differentiation. Poised chromatin states on specific targets could also be established by Ebf1 expression in T cells but not in NIH 3T3 cells, suggesting that Ebf1 acts as a "pioneer" factor in a hematopoietic chromatin context.

Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5.

Alternative lineage restriction and B cell fate commitment require the transcription factor Pax5, but the function of early B cell factor (EBF) in these processes remains mostly unexplored. Here we show that in the absence of EBF, 'expandable' and clonal lymphoid progenitor cells retained considerable myeloid potential. Conversely, ectopic expression of EBF in multipotential progenitor cells directed B cell generation at the expense of myeloid cell fates. EBF induced Pax5 and antagonized expression of genes encoding the transcription factors C/EBPalpha, PU.1 and Id2. Notably, sustained expression of EBF in Pax5-/- hematopoietic progenitor cells was sufficient to block their myeloid and T lineage potential in vivo. Furthermore, in Pax5-/- pro-B cells, higher EBF expression repressed alternative lineage genes. Thus, EBF can restrict alternative lineage 'choice' and promote commitment to the B cell fate independently of Pax5.