Zbigniew Dominski

Selection of splice sites in pre-mRNAs with short internal exons.

Model pre-mRNAs containing two introns and three exons, derived from the human beta-globin gene, were used to study the effects of internal exon length on splice site selection. Splicing was assayed in vitro in HeLa nuclear extracts and in vivo during transient expression in transfected HeLa cells. For substrates with internal exons 87, 104, and 171 nucleotides in length, in vitro splicing proceeded via a regular splicing pathway, in which all three exons were included in the spliced product. Primary transcripts with internal exons containing 23, 29, and 33 nucleotides were spliced by an alternative pathway, in which the first exon was joined directly to the third one. The internal exon was missing from the spliced product and together with two flanking introns was included in a large lariat structure. The same patterns of splicing were retained when transcripts containing 171-, 33-, and 29-nucleotide-long internal exons were spliced in vivo. A transcript containing a 51-nucleotide-long exon was spliced in vitro via both pathways but in vivo generated only a correctly spliced product. Skipping of short internal exons was reversed both in vitro and in vivo when purines in the upstream polypyrimidine tract were replaced by pyrimidines. The changes in the polypyrimidine tract achieved by these substitutions led in vitro to complete (transcripts containing 28 pyrimidines in a row) or partial (transcripts containing 15 pyrimidines in a row) restoration of a regular splicing pathway. Splicing in vivo of these transcripts led exclusively to the spliced product containing all three exons. These results suggest that a balance between the length of the uninterrupted polypyrimidine tract and the length of the exon is an important determinant of the relative strength of the splice sites, ensuring correct splicing patterns of multiintron pre-mRNAs.

Stem-Loop Binding Protein Facilitates 3′-End Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA

ABSTRACT The 3′ end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3′-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3′ processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP.

Nucleases of the Metallo-β-lactamase Family and Their Role in DNA and RNA Metabolism

ABSTRACT Proteins of the metallo-β-lactamase family with either demonstrated or predicted nuclease activity have been identified in a number of organisms ranging from bacteria to humans and has been shown to be important constituents of cellular metabolism. Nucleases of this family are believed to utilize a zinc-dependent mechanism in catalysis and function as 5′ to 3′ exonucleases and or endonucleases in such processes as 3′ end processing of RNA precursors, DNA repair, V(D)J recombination, and telomere maintenance. Examples of metallo-β-lactamase nucleases include CPSF-73, a known component of the cleavage/polyadenylation machinery, which functions as the endonuclease in 3′ end formation of both polyadenylated and histone mRNAs, and Artemis that opens DNA hairpins during V(D)J recombination. Mutations in two metallo-β-lactamase nucleases have been implicated in human diseases: tRNase Z required for 3′ processing of tRNA precursors has been linked to the familial form of prostate cancer, whereas inactivation of Artemis causes severe combined immunodeficiency (SCID). There is also a group of as yet uncharacterized proteins of this family in bacteria and archaea that based on sequence similarity to CPSF-73 are predicted to function as nucleases in RNA metabolism. This article reviews the cellular roles of nucleases of the metallo-β-lactamase family and the recent advances in studying these proteins.

Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

A CPSF-73 Homologue Is Required for Cell Cycle Progression but Not Cell Growth and Interacts with a Protein Having Features of CPSF-100

ABSTRACT Formation of the mature 3′ ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent β-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the β-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G1 phase, but surprisingly the arrested cells continue growing and reach the size typical of G2 cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3′ end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G1 progression and entry into S phase.

Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase

ABSTRACT The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3′ end of histone mRNAs. The protein that binds the 3′ end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.

Staged assembly of histone gene expression machinery at subnuclear foci in the abbreviated cell cycle of human embryonic stem cells

Human embryonic stem (hES) cells have an abbreviated G1 phase of the cell cycle. How cells expedite G1 events that are required for the initiation of S phase has not been resolved. One key regulatory pathway that controls G1/S-phase transition is the cyclin E/CDK2-dependent activation of the coactivator protein nuclear protein, ataxia–telangiectasia locus/histone nuclear factor-P (p220NPAT/HiNF-P) complex that induces histone gene transcription. In this study, we use the subnuclear organization of factors controlling histone gene expression to define mechanistic differences in the G1 phase of hES and somatic cells using in situ immunofluorescence microscopy and fluorescence in situ hybridization (FISH). We show that histone gene expression is supported by the staged assembly and modification of a unique subnuclear structure that coordinates initiation and processing of transcripts originating from histone gene loci. Our results demonstrate that regulatory complexes that mediate transcriptional initiation (e.g., p220NPAT) and 3′-end processing (e.g., Lsm10, Lsm11, and SLBP) of histone gene transcripts colocalize at histone gene loci in dedicated subnuclear foci (histone locus bodies) that are distinct from Cajal bodies. Although appearance of CDK2-phosphorylated p220NPAT in these domains occurs at the time of S-phase entry, histone locus bodies are formed ≈1 to 2 h before S phase in embryonic cells but 6 h before S phase in somatic cells. These temporal differences in the formation of histone locus bodies suggest that the G1 phase of the cell cycle in hES cells is abbreviated in part by contraction of late G1.

Structure of histone mRNA stem-loop, human stem-loop binding protein and 3′hExo ternary complex

Recognizing a Stem-Loop Structure Metazoan histone messenger RNAs (mRNAs) have a conserved stem-loop (SL) structure at their 3′-end. The stem-loop is bound by the stem-loop binding protein (SLBP), which is required for histone mRNA 3′-end processing, export, stability, and translation. The 3′-5′ exonuclease 3′hExo also binds the SL and trims off three nucleotides. Tan et al. (p. 318) determined the high-resolution structure of the SL bound by the RNA-binding domain (RBD) of human SLBP together with human 3′hExo. The conformation of the loop differed substantially from other RNA tetraloops and the SLBP RBD may function as a ruler that can measure the length of the stem. Although the SLBP directly recognizes the guanine base of the second nucleotide of the stem, it appears that SLBP and 3′hExo recognize the unique shape of the SL. Two RNA binding proteins recognize their target RNA through shape, rather than sequence. Metazoan replication-dependent histone messenger RNAs (mRNAs) have a conserved stem-loop (SL) at their 3′-end. The stem-loop binding protein (SLBP) specifically recognizes the SL to regulate histone mRNA metabolism, and the 3′-5′ exonuclease 3′hExo trims its 3′-end after processing. We report the crystal structure of a ternary complex of human SLBP RNA binding domain, human 3′hExo, and a 26-nucleotide SL RNA. Only one base of the SL is recognized specifically by SLBP, and the two proteins primarily recognize the shape of the RNA. SLBP and 3′hExo have no direct contact with each other, and induced structural changes in the loop of the SL mediate their cooperative binding. The 3′ flanking sequence is positioned in the 3′hExo active site, but the ternary complex limits the extent of trimming.

Reprogramming the pluripotent cell cycle: restoration of an abbreviated G1 phase in human induced pluripotent stem (iPS) cells.

Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are reprogrammed to possess stem cell like properties. However, the extent to which iPS cells exhibit unique properties of the human embryonic stem (hES) cell cycle remains to be established. hES cells are characterized by an abbreviated G1 phase (∼2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e., histone locus bodies (HLBs)]. We therefore examined cell cycle parameters of iPS cells in comparison to hES cells. Analysis of DNA synthesis [5‐bromo‐2′‐deoxy‐uridine (BrdU) incorporation], cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immunofluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase (∼2.5 h) and an abbreviated cell cycle (16–18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within 1.5–2 h). Thus, reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency. J. Cell. Physiol. 226: 1149–1156, 2011.

Studies of the 5' exonuclease and endonuclease activities of CPSF-73 in histone pre-mRNA processing.

ABSTRACT Processing of histone pre-mRNA requires a single 3′ endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2′-O-methyl nucleotide and occurs in the 5′-to-3′ direction. The U7-dependent 5′ exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5′-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5′ exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5′ exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.