U7 deciphered: the mechanism that forms the unusual 3′ end of metazoan replication-dependent histone mRNAs

In animal cells, replication-dependent histone mRNAs end with a highly conserved stem–loop structure followed by a 4- to 5-nucleotide single-stranded tail. This unique 3′ end distinguishes replication-dependent histone mRNAs from all other eukaryotic mRNAs, which end with a poly(A) tail produced by the canonical 3′-end processing mechanism of cleavage and polyadenylation. The pioneering studies of Max Birnstiel's group demonstrated nearly 40 years ago that the unique 3′ end of animal replication-dependent histone mRNAs is generated by a distinct processing mechanism, whereby histone mRNA precursors are cleaved downstream of the stem–loop, but this cleavage is not followed by polyadenylation. The key role is played by the U7 snRNP, a complex of a ∼60 nucleotide U7 snRNA and many proteins. Some of these proteins, including the enzymatic component CPSF73, are shared with the canonical cleavage and polyadenylation machinery, justifying the view that the two metazoan pre-mRNA 3′-end processing mechanisms have a common evolutionary origin. The studies on U7 snRNP culminated in the recent breakthrough of reconstituting an entirely recombinant human machinery that is capable of accurately cleaving histone pre-mRNAs, and determining its structure in complex with a pre-mRNA substrate (with 13 proteins and two RNAs) that is poised for the cleavage reaction. The structure uncovered an unanticipated network of interactions within the U7 snRNP and a remarkable mechanism of activating catalytically dormant CPSF73 for the cleavage. This work provides a conceptual framework for understanding other eukaryotic 3′-end processing machineries.

Slaying the last unicorn: discovery of histones in the microalga Nanochlorum eucaryotum

Histones are the principal constituents of eukaryotic chromatin. The four core histones (H2A, H2B, H3 and H4) are conserved across sequenced eukaryotic genomes and therefore thought to be universal to eukaryotes. In the early 1980s, however, a series of biochemical investigations failed to find evidence for histones or nucleosomal structures in the microscopic green alga Nanochlorum eucaryotum . If true, derived histone loss in this lineage would constitute an exceptional case that might help us further understand the principles governing eukaryotic gene regulation. To substantiate these earlier reports of histone loss in N. eucaryotum, we sequenced, assembled and quantified its transcriptome. Following a systematic search for histone-fold domains in the assembled transcriptome, we detect orthologues to all four core histones. We also find histone mRNAs to be highly expressed, comparable to the situation in other eukaryotes. Finally, we obtain characteristic protection patterns when N. eucaryotum chromatin is subjected to micrococcal nuclease digestion, indicating widespread formation of nucleosomal complexes in vivo . We conclude that previous reports of missing histones in N. eucaryotum were mistaken. By all indications, Nanochlorum eucaryotum has histone-based chromatin characteristic of most eukaryotes.

A region of SLBP outside the mRNA processing domain is essential for deposition of histone mRNA into the Drosophila egg

Replication-dependent histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending in a stemloop instead of a polyA tail, and are normally regulated coordinately with DNA replication. SLBP binds the 3’ end of histone mRNA, and is required for processing and translation. During Drosophila oogenesis, large amounts of histone mRNAs and proteins are deposited in the developing oocyte.The maternally deposited histone mRNA is synthesized in stage 10B oocytes after the nurse cells complete endoreduplication. We report that in WT stage 10B oocytes, the Histone Locus Bodies (HLBs), formed on the histone genes, produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Two mutants of SLBP, one with reduced expression and another with a 10 aa deletion, fail to deposit sufficient histone mRNA in the oocyte, and don't transcribe the histone genes in stage 10B. Mutations in a putative SLBP nuclear localization sequence overlapping the deletion, phenocopy the deletion. We conclude a high concentration of SLBP in the nucleus of stage 10B oocytes is essential for histone gene transcription.

Slaying the last unicorn - discovery of histones in the microalga Nanochlorum eucaryotum

ABSTRACTHistones are the principal constituents of eukaryotic chromatin. The four core histones (H2A, H2B, H3, and H4) are conserved across sequenced eukaryotic genomes and therefore thought to be universal to eukaryotes. In the early 1980s, however, a series of biochemical investigations failed to find evidence for histones or nucleosomal structures in the microscopic green alga Nanochlorum eucaryotum. If true, derived histone loss in this lineage would constitute an exceptional case that might help us further understand the principles governing eukaryotic gene regulation. To substantiate these earlier reports of histone loss in N. eucaryotum, we sequenced, assembled and quantified its transcriptome. Following a systematic search for histone-fold domains in the assembled transcriptome, we detect orthologs to all four core histones. We also find histone mRNAs to be highly expressed, comparable to the situation in other eukaryotes. Finally, we obtain characteristic protection patterns when N. eucaryotum chromatin is subjected to micrococcal nuclease digestion, indicating widespread formation of nucleosomal complexes in vivo. We conclude that previous reports of missing histones in N. eucaryotum were mistaken. By all indications, N. eucaryotum has histone-based chromatin characteristic of most eukaryotes.

piRNAs prevent runaway amplification of siRNAs from ribosomal RNAs and histone mRNAs

ABSTRACTPiwi-interacting RNAs (piRNAs) are a largely germline-specific class of small RNAs found in animals. Although piRNAs are best known for silencing transposons, they regulate many different biological processes. Here we identify a role for piRNAs in preventing runaway amplification of small interfering RNAs (siRNAs) from certain genes, including ribosomal RNAs (rRNAs) and histone mRNAs. In Caenorhabditis elegans, rRNAs and some histone mRNAs are heavily targeted by piRNAs, which facilitates their entry into an endogenous RNA interference (RNAi) pathway involving a class of siRNAs called 22G-RNAs. Under normal conditions, rRNAs and histone mRNAs produce relatively low levels of 22G-RNAs. But if piRNAs are lost, 22G-RNA production is highly elevated. We show that 22G-RNAs produced downstream of piRNAs likely function in a feed-forward amplification circuit. Thus, our results suggest that piRNAs facilitate low-level 22G-RNA production while simultaneously obstructing the 22G-RNA machinery to prevent runaway amplification from certain RNAs. Histone mRNAs and rRNAs are unique from other cellular RNAs in lacking polyA tails, which may promote feed-forward amplification of 22G-RNAs. In support of this, we show that the subset of histone mRNAs that contain polyA tails are largely resistant to silencing in piRNA mutants.

A region of Drosophila SLBP distinct from the histone pre-mRNA binding and processing domains is essential for deposition of histone mRNA in the oocyte

ABSTRACTDuring Drosophila oogenesis, large amounts of histone mRNA and proteins are deposited in the developing oocyte. These are sufficient for the first 14 embryonic cell cycles and provide the developing embryo with sufficient histone proteins until the zygotic histone genes are activated. The maternally deposited histone mRNA is synthesized in stage 10b of oogenesis after completion of endoreduplication of the nurse cells. Histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending instead in a conserved stemloop instead of a polyA tail. The Stem-loop binding protein (SLBP) binds the 3’ end of histone mRNA and is essential for both the biosynthesis and translation of histone mRNA. We report that a 10 aa region in SLBP, which is not required for processing in vitro, is essential for transcription of histone mRNA in the stage 10b oocyte. In stage 10b the Histone Locus Bodies (HLBs) produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Mutants expressing this SLBP develop normally, produce small amounts of polyadenylated histone mRNA throughout development, but little histone mRNA in stage 10b resulting in death of the embryos in the first hr of development.

Loss of Histone Locus Bodies in the Mature Hemocytes of Larval Lymph Gland Result in Hyperplasia of the Tissue in mxc Mutants of Drosophila

Mutations in the multi sex combs (mxc) gene in Drosophila results in malignant hyperplasia in larval hematopoietic tissues, called lymph glands (LG). mxc encodes a component of the histone locus body (HLB) that is essential for cell cycle-dependent transcription and processing of histone mRNAs. The mammalian nuclear protein ataxia-telangiectasia (NPAT) gene, encoded by the responsible gene for ataxia telangiectasia, is a functional Mxc orthologue. However, their roles in tumorigenesis are unclear. Genetic analyses of the mxc mutants and larvae having LG-specific depletion revealed that a reduced activity of the gene resulted in the hyperplasia, which is caused by hyper-proliferation of immature LG cells. The depletion of mxc in mature hemocytes of the LG resulted in the hyperplasia. Furthermore, the inhibition of HLB formation was required for LG hyperplasia. In the mutant larvae, the total mRNA levels of the five canonical histones decreased, and abnormal forms of polyadenylated histone mRNAs, detected rarely in normal larvae, were generated. The ectopic expression of the polyadenylated mRNAs was sufficient for the reproduction of the hyperplasia. The loss of HLB function, especially 3′-end processing of histone mRNAs, is critical for malignant LG hyperplasia in this leukemia model in Drosophila. We propose that mxc is involved in the activation to induce adenosine deaminase-related growth factor A (Adgf-A), which suppresses immature cell proliferation in LG.

Widespread roles for piRNAs and WAGO-class siRNAs in shaping the germline transcriptome of Caenorhabditis elegans

Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.