Adapted from Reactome, see: [http://www.reactome.org/cgi-bin/eventbrowser_st_id?ST_ID=REACT_1675.1 REACT 1675.1] This process describes the conversion of precursor messenger RNA into mature messenger RNA (mRNA). The pre-mRNA molecule undergoes three main modifications. These modifications are 5' capping, 3' polyadenylation, and RNA splicing, which occur in the cell nucleus before the RNA is translated. 5' Capping: Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5' end. The cap protects the 5' end of the primary RNA transcript from attack by ribonucleases that have specificity to the 3'5' phosphodiester bonds. 3' Processing: The pre-mRNA processing at the 3' end of the RNA molecule involves cleavage of its 3' end and then the addition of about 200 adenine residues to form a poly(A) tail. As the poly(A) tails is synthesised, it binds multiple copies of poly(A) binding protein, which protects the 3'end from ribonuclease digestion. Splicing: RNA splicing is the process by which introns, regions of RNA that do not code for protein, are removed from the pre-mRNA and the remaining exons connected to re-form a single continuous molecule. Description adapted from Wikipedia: http://en.wikipedia.org/wiki/Post-transcriptional_modification Gene lists from obtained from InterPro 9-23-2003: Mm RNA-binding region RNP-1 (RNA recognition motif) Adapted from http://www.reactome.org Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro GenBank sequence: K00027 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro PMID: 14729963 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro inferred from: SURP 2, G patch protein, UniProt:O60378 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro PMID: 12466556 Derived from InterPro PMID: 12738786, PMID: 12024014 (associated with hnRNP A1 splicing) Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro GenBank sequence: M10328, M18004 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro GenBank sequence: M10329 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Antagonist splicing factor Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro GenBank sequence: J00645 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro PMID: 12773558 Derived from InterPro Derived from InterPro GenBank sequence: AB021173 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived 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InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro PMID: 12773558 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro FUSI MOUSE PMID: 11684676 Derived from InterPro Derived from InterPro PMID: 14729963 Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived 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SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection. Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Derived from InterPro Endonucleolytic cleavage separates the pre-mRNA into an upstream fragment destined to become the mature mRNA, and a downstream fragment that is rapidly degraded. Cleavage depends on two signals in the RNA, a highly conserved hexanucleotide, AAUAAA, 10 to 30 nucleotides upstream of the cleavage site, and a poorly conserved GU- or U-rich downstream element. Additional sequences, often upstream of AAUAAA, can enhance the efficiency of the reaction. Cleavage occurs most often after a CA dinucleotide. A single gene can have more than one 3’ processing site. Cleavage is preceded by the assembly of a large processing complex, the composition of which is poorly defined. ATP, but not its hydrolysis, is required for assembly. Cleavage at the 3’-end of mRNAs depends on a number of protein factors. CPSF, a heterotetramer, binds specifically to the AAUAAA sequence. The heterotrimer CstF binds the downstream element. CF I, which appears to be composed of two subunits, one of several related larger polypeptides and a common smaller one, also binds RNA, but with unknown specificity. RNA recognition by these proteins is cooperative. Cleavage also requires CF II, composed of at least two subunits, and poly(A) polymerase, the enzyme synthesizing the poly(A) tail in the second step of the reaction. The polypeptide catalyzing the hydrolysis of the phosphodiester bond remains to be identified. Cleavage produces a 3’-OH on the upstream fragment and a 5’-phosphate on the downstream fragment. At some unknown point after cleavage, the downstream RNA fragment, CstF, CF I and CF II are thought to be released, whereas CPSF and poly(A) polymerase remain to carry out polyadenylation. In addition to the methylation of the 5’-cap, there is methylation of internal nucleotides in the mRNA. This methylation can occur in translated and untranslated regions. One to three methyl groups have been seen per mRNA molecule, but methylation is non-stoichiometric. The most frequent methylation observed is at the N6 position of adenosine. The function of mRNA internal methylation, if any, is unknown. Krainer, A, 2003-06-05 The active C complex is formed due to a conformational change in the intermediate C complex. After formation of the active C complex, the splicing reactions occur very rapidly. Krainer, A, 2003-06-05 inorganic phosphate Cis-regulatory nucleotide splicing motifs, regulated by the binding of SR-proteins. Include exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs. The process in which excision of introns from the primary transcript of messenger RNA (mRNA) is followed by ligation of the two exon termini exposed by removal of each intron, is called mRNA splicing. Most of the mRNA is spliced by the major pathway, involving the U1, U2, U4, U5 and U6 snRNPs. A minor fraction, about 1 %, of the mRNAs are spliced via the U12 dependent pathway. Krainer, A, 2003-06-05 Clk proteins can interact with and phosphorylate SR proteins. These proteins are capable of autophosphorylation and may alter the subnuclear localization of SR proteins. While the Clk proteins phosphorylate RS domains, they themselves contain RS-like domains and are also known to be autophosphorylated on tyrosines. Phosphorylates RS domains The formation of the B complex is ATP-dependent, and both the 5’ and 3’ splice sites are essential for B complex assembly. The U4 and U6 snRNPS are extensively base-paired, and this U4:U6 complex associates with the U5 snRNP to form a tri-snRNP particle. This tri-snRNP particle then binds to the spliceosomal A complex, to form the spliceosomal B complex. Krainer, A, 2003-06-05 After the nascent pre-mRNA undergoes the initial capping and methylation reactions, it gets associated with numerous factors, including the various heterogeneous nuclear ribonucleoproteins (hnRNPS), the nuclear Cap-Binding Complex, and many splicing factors that make the pre-mRNA a substrate for splicing, 3’-end processing, and in some cases editing. Krainer, A, 2003-06-05 The upstream fragment generated by 3’ cleavage of the pre-mRNA receives a poly(A) tail of approximately 250 AMP residues in a reaction depending on the AAUAAA sequence 10 to 30 nucleotides upstream of the 3’ end. Polyadenylation is carried out by three proteins: Poly(A) polymerase carries the catalytic activity. The enzyme has no specificity for any particular RNA sequence, and it also has a very low affinity for the RNA. Under physiological conditions, the activity of poly(A) polymerase thus depends on two auxiliary factors, both of which bind to specific RNA sequences and recruit the enzyme by a direct contact. One of these proteins is the heterotetrameric CPSF, which binds the AAUAAA sequence and is also essential for 3’ cleavage. The second is the nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once this has reached a length of about ten nucleotides. Stimulation of poly(A) polymerase by both proteins is synergistic and results in processive elongation of the RNA, i.e. the polymerase adds AMP residues without dissociating from the RNA. The processive reaction is terminated when the tail has reached a length of about 250 nucleotides. Cleavage produces a 3’-OH on the upstream fragment and a 5’-phosphate on the downstream fragment. At some unknown point after cleavage, the downstream RNA fragment, CstF, CF I and CF II are thought to be released, whereas CPSF and poly(A) polymerase remain to carry out polyadenylation. The spliceosomal C complex is a very short-lived intermediate; the splicing intermediates are rapidly converted to splicing products. Also, the spliced products are released very rapidly, and no complex containing both the splicing products has been isolated. Conversion of the spliceosomal B complex to the spliceosomal C complex requires ATP. The extensive base-pairing between the U4 and U6 snRNAs is disrupted during the formation of the C complex, which is thought to require helicase-type activity associated with the DEAD box factors. Krainer, A, 2003-06-05 The second step of the splicing reaction results in cleavage of the transcript at the 3’splice site, and results in ligation of the two exons and excision of the intron. Krainer, A, 2003-06-05 Phosphorylates RS domains Clk proteins can interact with and phosphorylate SR proteins. These proteins are capable of autophosphorylation and may alter the subnuclear localization of SR proteins. While the Clk proteins phosphorylate RS domains, they themselves contain RS-like domains and are also known to be autophosphorylated on tyrosines. RS dipeptides Any covalent change in a primary (nascent) mRNA transcript is mRNA Processing. For successful gene expression, the primary mRNA transcript needs to be converted to a mature mRNA prior to its translation into polypeptide. Eucaryotic mRNAs undergo a series of complex processing reactions; these begin on nascent transcripts as soon as a few ribonucleotides have been synthesized during transcription by RNA Polymerase II, through the export of the mature mRNA to the cytoplasm, and culminate with mRNA turnover in the cytoplasm. Carmichael, GG, Hammarskjold, ML, Hastings, M, Krainer, A, Marzluff, WF, 2003-06-05 The 5’-ends of all eukaryotic pre-mRNAs studied thus far are converted to cap structures. The cap is thought to influence splicing of the first intron, and is bound by ’cap-binding’ proteins, CBP80 and CBP20, in the nucleus. The cap is important for translation initiation, and it also interacts with the poly(A)terminus, via proteins, resulting in circularization of the mRNA to facilitate multiple rounds of translation. The cap is also important for mRNA stability, protecting it from 5’ to 3’ nucleases, and is required for mRNA export to the cytoplasm. The capping reaction usually occurs very rapidly on nascent transcripts; after the synthesis of only a few nucleotides by RNA polymerase II. The capping reaction involves the conversion of the 5’-end of the nascent transcript from a triphosphate to a diphosphate by a RNA 5’-triphosphatase, followed by the addition of a guanosine monophosphate by the mRNA guanylyltransferase, to form a 5’-5’-triphosphate linkage. This cap is then methylated by 2’-O-methyltransferases. Buratowski, S, 2003-10-15 Pre-mRNA transcripts become rapidly associated with many RNA-binding proteins, including hnRNP proteins, cap-binding proteins, SR proteins, etc; in the test tube this binding does not require splice sites or ATP. The E complex, or early complex, is the first detectable functional intermediate in spliceosome assembly in vitro. It is an ATP-independent complex. When a functional 5? splice site is present, it is bound by the U1 snRNP. The splicing factor U2AF (65 and 35 kDa subunits) binds to the polypyrimidine tract (Y)n and the AG dinucleotide at the 3’ splice site, respectively. SF1/mBBP binds to the branch site. Binding of many of these factors is cooperative; e.g., SR proteins and U2AF apparently interact with each other, facilitating their binding to the pre-mRNA.In the presence of ATP, the E complex is converted to the first ATP-dependent spliceosomal complex, namely the A complex. Krainer, A, 2003-06-05 In the first catalytic step of mRNA splicing, the 2’ OH group of the bulged A at the branch site performs a nucleophilic attack on the 5’ splice site phosphodiester bond, resulting in cleavage of the bond between the 5’ exon and the 5’ end of the intron, and formation of a new bond between the 5’ end of the intron and the branch site A. This results in a lariat-shaped intermediate, with the intron still attached to the 3’ exon. The branch site A has a 2’-5’ phosphodiester bond with the G at the beginning of the intron, in addition to the usual 5’-3’ and 3’-5’phosphodiester bonds. Krainer, A, 2003-06-05 The A complex is the first ATP-dependent complex in spliceosome assembly. U2AF recruits the U2 snRNP to bind to the branch site in the E complex in an ATP-dependent fashion, to form the A complex. The U2 snRNA base-pairs with the branch site, causing the branch-site adenosine to bulge out, which later positions it for nucleophillic attack at the 5’ splice site. The A complex serves as a substrate for formation of the B complex. Krainer, A, 2003-06-05 The 3’ ends of eukaryotic mRNAs are generated by posttranscriptional processing of an extended primary transcript. For almost all RNAs, 3’-end processing consists of two steps: (i) the mRNA is first cleaved at a particular phosphodiester bond downstream of the coding sequence, (ii) the upstream fragment then receives a poly(A) tail of approximately 250 adenylate residues, whereas the downstream fragment is degraded. The two partial reactions are coupled so that reaction intermediates are usually undetectable. While 3’ processing can be studied as an isolated event in vitro, it appears to be connected to transcription, splicing, and transcription termination in vivo. The only known exception to the rule of cleavage followed by polyadenylation are the major histone mRNAs, which are cleaved but not polyadenylated. spliceosome pathway PW:0001073 Pathway Ontology