A 3' splice site-binding sequence in the catalytic core of a group I intron

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.     

Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.     

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.     

Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.     

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.     

Functional recognition of the 3' splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.     

Identification of a factor that links apoptotic cells to phagocytes

Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif--it bound particularly strongly to cells expressing alpha(v)beta(3) integrin. The NIH3T3 cell transformants that expressed a high level of alpha(v)beta(3) integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.     

Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5' cap

Eukaryotic cellular mRNAs have a 5' cap structure (m7 GpppX) that facilitates binding to ribosomes and is required for efficient translation. A specific initiation factor, eIF-4F, mediates the function of the cap and consists of three subunits, one of which, eIF-4E, binds the cap. This subunit is present in limiting amounts in the cell, and is thought to be regulated by phosphorylation: decreased phosphorylation of eIF-4E following various treatments correlates with a decrease in cellular translation rate. These observations suggest that eIF-4E lies on the mitogenic signal transduction pathway, and we reasoned that overexpression of eIF-4E might profoundly affect cellular growth properties. We report here that overexpression of eIF-4E in NIH 3T3 and Rat 2 fibroblasts causes their tumorigenic transformation as determined by three criteria: formation of transformed foci on a monolayer of cells; anchorage-independent growth; and tumour formation in nude mice.     

Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA

The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.     

关于一类有界拟凸域全纯自同构群的一个注记

证明了从拟凸域Ｅ（ｍ，ｎ，ｋ）＝（ｚ，ｗ）∈Ｃ＾ｍ＋ｍ；／ｚ／＾２＋／ｗ／＾２ｋ〉１，ｚ∈Ｃ＾ｎ，ｗ∈Ｃ＾ｍ，ｋ〉０／的作地一不变Ｋａｈｌｅｒ度量都可以导出相贩Ａｕｔ（Ｅ）。     

MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8.

Interactions between major histocompatibility complex (MHC) molecules and the CD4 or CD8 coreceptors have a major role in intrathymic T-cell selection. On mature T cells, each of these two glycoproteins is associated with a class-specific bias in MHC molecule recognition by the T-cell receptor. CD4+ T cells respond to antigen in association with MHC class II molecules and CD8+ T cells respond to antigen in association with MHC class I molecules. Physical interaction between the CD4/MHC class II molecules and CD8/MHC class I molecules has been demonstrated by cell adhesion assay, and a binding site for CD8 on class I has been identified. Here we demonstrate that a region of the MHC class II beta-chain beta 2 domain, structurally analogous to the CD8-binding loop in the MHC class I alpha 3 domain, is critical for function with both mouse and human CD4.     

Constitutive expression of (2'-5') oligo A synthetase confers resistance to picornavirus infection

Study of the mechanisms by which interferon (IFN) treatment of cells induces resistance to virus infections has been complicated by the multiple biochemical changes induced. Over 20 proteins are increased by IFN, including the double-stranded (ds) RNA-activated protein kinase, (2'-5') oligo A synthetase, surface proteins such as the major histocompatibility complex (MHC) proteins, and various proteins with unknown functions. The availability of cloned complementary DNAs for several IFN-induced proteins now allows us to probe their roles in IFN action. For instance, the murine Mx protein has been shown to confer resistance, to influenza virus. We studied chinese hamster ovary (CHO) cell clones expressing high constitutive levels of (2'-5') A synthetase as a result of transfection with the cDNA encoding the enzyme form which has a relative molecular mass (Mr) of 40K. Elevated enzyme correlates directly with resistance to infection by a picornavirus such as Mengo, but does not make the cells resistant to vesicular stomatitis virus (VSV).     

A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules

Class II and class I histocompatibility molecules allow T cells to recognize 'processed' polypeptide antigens. The two polypeptide chains of class II molecules, alpha and beta, are each composed of two domains (for review see ref. 6); the N-terminal domains of each, alpha 1 and beta 1, are highly polymorphic and appear responsible for binding peptides at what appears to be a single site and for being recognized by MHC-restricted antigen-specific T cells. Recently, the three-dimensional structure of the foreign antigen binding site of a class I histocompatibility antigen has been described. Because a crystal structure of a class II molecule is not available, we have sought evidence in class II molecules for the structural features observed in the class I binding site by comparing the patterns of conserved and polymorphic residues of twenty-six class I and fifty-four class II amino acid sequences. The hypothetical class II foreign-antigen binding site we present is consistent with mutation experiments and provides a structural framework for proposing peptide binding models to help understand recent peptide binding data.     

Internal initiation of translation mediated by the 5' leader of a cellular mRNA.

A Robosome-scanning model has been proposed to explain the initiation of eukaryotic messenger RNAs in which binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap-structure at the end of the mRNA and the cap-binding protein complex eIF-4F. But picornaviral mRNAs do not have a 5' terminal cap structure and are translated by internal ribosome binding. A cellular mRNA, encoding the immunoglobulin heavy-chain binding protein, can be translated in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNAs is inhibited. We report here that the 5' leader of the binding protein mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, indicating that translation initiation by an internal ribosome-binding mechanism is used by eukaryotic mRNAs.