3′ splice site recognition in nematode trans-splicing involves enhancer- dependent recruitment of U2 snRNP

Romfo, Charles M.; Maroney, Patricia A.; Wu, Shenping; Nilsen, Timothy W.

doi:10.1017/s1355838201010263

Cited by 15 publications

(22 citation statements)

References 27 publications

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“…Alternatively (or additionally), an important role of ESEs in normal extracts could indeed be to facilitate U2AF recruitment, and results from a number of laboratories and different pre-mRNA substrates are certainly consistent with this (5,14,37,47). Also consistent are the results presented in Fig.…”

Section: Discussionmentioning

confidence: 70%

“…One type of sequences bound by SR proteins are purine-rich exonic splicing enhancers (ESE), which stimulate splicing of pre-mRNAs containing weak 3Ј splice sites (reviewed in references 7 and 42). Based upon experiments using purified components, Zuo and Maniatis (60) proposed that SR proteins bound to ESEs facilitate recruitment of U2AF 65 to the Py tract via bridging interactions mediated by U2AF 35 (4,13,14,37). Other results, however, argued that U2AF 65 recruitment was not the rate-limiting step in ESE-dependent splicing (20,26).…”

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confidence: 99%

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Dual Function for U2AF³⁵ in AG-Dependent Pre-mRNA Splicing

Guth¹,

Tange²,

Kellenberger

et al. 2001

Molecular and Cellular Biology

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The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF 65 ) binds to the polypyrimidine (Py) tract preceding the 3 splice site, while the 35-kDa subunit (U2AF 35 Intron removal from mRNA precursors (pre-mRNA splicing) is an essential step of gene expression in eukaryotes. The precise recognition of the intron boundaries, the 5Ј and 3Ј splice sites, is achieved by small nuclear RNPs (snRNPs) and non-snRNP proteins. The 5Ј splice site is initially recognized by U1 snRNP, and the 3Ј splice site region is recognized by U2 snRNP. Subsequent addition of the U4/U6/U5 tri-snRNP forms the spliceosome, the macromolecular complex within which splicing catalysis takes place (reviewed in references 6 and 23).Several sequence elements help to define the 3Ј splice site region in higher eukaryotes (reviewed in reference 35) : the branchpoint (BP) sequence, usually followed by a pyrimidinerich sequence (the polypyrimidine tract or Py tract), and a conserved AG dinucleotide at the 3Ј end of the intron. The BP contains an adenosine residue that forms a 2Ј to 5Ј phosphodiester bond with the 5Ј end of the intron during the first catalytic step of the splicing reaction (39). U2 snRNP binds to the BP through base pairing interactions between this sequence and U2 snRNA (31,33,50,56). U2 snRNP binding requires auxiliary factors, including SF1/mBBP and U2AF (22,24,40). SF1/mBBP has been shown to specifically recognize the BP (2, 34) and play a kinetic role in spliceosome assembly (17, 41). U2AF is a heterodimer of 65 and 35-kDa subunits (52). U2AF 65 binds specifically to the Py tract via its RNA recognition motifs (RRMs) (53) and contacts the BP via its RS domain (11, 44), whereas U2AF 35 contacts the AG dinucleotide at the 3Ј splice site (30,51,58).The 3Ј splice site AG marks the 3Ј intron boundary and is involved in exon ligation, the second catalytic step of the splicing reaction. For some AG-dependent substrates, however, this dinucleotide is already required for early steps of spliceosome assembly prior to catalysis (36). AG-dependent substrates typically contain weak Py tracts, and substrates with strong Py tracts generally do not require the presence of the 3Ј splice site AG before the second catalytic step and are considered AG independent. Interaction between U2AF35 and the 3Ј splice site AG dinucleotide was shown to stabilize U2AF 65 binding to a weak Py tract and to be essential for splicing of AG-dependent substrates (51).An alternative set of interactions has been proposed for U2AF 35 . The arginine-serine (RS) region of U2AF 35 has been shown to establish protein-protein interactions with splicing factors of the SR family (49) (reviewed in references 10, 13, 29, and 45). One type of sequences bound by SR proteins are purine-rich exonic splicing enhancers (ESE), which stimulate splicing of pre-mRNAs containing weak 3Ј splice sites (reviewed in references 7 and 42). Based upon experiments using

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Section: Discussionmentioning

confidence: 70%

mentioning

confidence: 99%

Dual Function for U2AF³⁵ in AG-Dependent Pre-mRNA Splicing

Guth¹,

Tange²,

Kellenberger

et al. 2001

Molecular and Cellular Biology

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“…For reactions in which the U2 snRNP was blocked, the reaction mixtures included 200 ng of a 2Ј-OMe oligonucleotide complementary to nt 29 to 45 (branch point interaction region). Nuclease protection assays were carried out essentially as previously described (32,40), except that 420 U of micrococcal nuclease (Worthington) was used and the digestion occurred on ice for 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…The RNA binding domain is involved in the interaction of SR proteins with elements within the pre-mRNA substrate termed splicing enhancers. Splicing enhancers serve as a platform for SR proteins to recruit essential factors to the 3Ј splice site (17,40,62). In addition, splicing enhancers can counteract the negative regulation of splicing imposed by splicing silencers (26).…”

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confidence: 99%

Multiple Roles for SR Proteins intransSplicing

Furuyama

Bruzik

2002

Molecular and Cellular Biology

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The trans-splicing reaction involves the association of 5 and 3 splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3 acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5 splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.trans splicing involves a productive interaction between 5Ј and 3Ј splice sites present on separate transcripts. In nematodes, the trans-spliced 5Ј splice site is contained on a unique small RNA (spliced leader RNA [SL RNA]) that harbors both the donated exon and a region exhibiting snRNP-like characteristics. The SL RNP contains Sm proteins, which are common to other U snRNPs, as well as specific proteins in addition to a trimethylguanosine cap (for a review, see reference 38). This unusual situation, where the 5Ј exon and an RNP component of the reaction are present in a single molecule, highlights the most apparent difference between cis splicing and trans splicing (5, 51, 52). Required RNP components of a trans spliceosome include the SL, U2, and U4/U6.U5 snRNPs but, notably, not U1 snRNP (33). Several lines of evidence have demonstrated that the 5Ј end of U1, which normally pairs with the 5Ј splice site in cis splicing, is not required for trans splicing (22). Recent work has demonstrated, however, that U1 snRNP can exert a large effect on the efficiency of trans splicing via exon definition-like interactions (2). In addition, there are interactions between several of the snRNPs involved in trans splicing that are not present in a cis spliceosome. These include an interaction between the SL RNP and U6 (57) as well as contact between the exonic portion of the SL RNP and U5, even in the absence of a trans-acceptor substrate (33). These interactions, coupled with the fact that the splice sites are contained on separate RNAs, have led to speculation as to how the splice sites might be initially juxtaposed in a forming trans spliceosome, prior to any extensive RNA-RNA rearrangements that would precede catalysis (3).The role(s) of protein factors in authentic trans splicing has been examined much less thoroughly. However, the serine/ arginine-rich non-snRNP spl...

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“…A polypyrimide tract is observed upstream of the AG dinucleotides in kinetoplastids (Liang et al 2003), cnidarians and in C. intestinalis (Vandenberghe et al 2001), whereas in nematodes there is no polypyrimidine tract associated to the 3' splice site. In nematodes a conserved sequence UUUCAG/ (AG, acceptor site) is required for proper processing (Conrad et al 1993, Romfo et al 2001. Finally, in both processes it is possible to find out the participation of ESEs, SR proteins as well as similar spliceosome which are formed by almost the same snRNPs and proteins (Sanford & Bruzik 1999, Liang et al 2003.…”

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confidence: 99%

Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

Mayer

Flöeter-Winter²

2005

Mem. Inst. Oswaldo Cruz

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Since the discovery that genes are split into intron and exons, the studies of the mechanisms involved in splicing pointed to presence of consensus signals in an attempt to generalize the process for all living cells. However, as discussed in the present review, splicing is a theme full of variations. The trans-splicing of pre-mRNAs, the joining of exons from distinct transcripts, is one of these variations with broad distribution in the phylogenetic tree. The biological meaning of this phenomenon is discussed encompassing reactions resembling a possible noise to mechanisms of gene expression regulation. All of them however, can contribute to the generation of life diversity.Key words: RNA processing -alternative trans-splicing -spliced leader RNA -trypanosomatids -nematodes -mammalian Most of the protein-coding eukaryotic genes display an interrupted structure alternating exons and introns. After transcription, introns must be removed from the primary transcript (pre-mRNA) to generate a translatable mature mRNA. It is interesting to note that the mature mRNA is constituted of 5' and 3' untranslatable regions (UTR) flanking the open reading frame (ORF) and that UTR are also exons. The precise excision of the introns and the joining of neighboring exons is a complex process generally named splicing, and when this processing occurs within a single pre-mRNA molecule it can also be called cis-splicing (Moore et al. 1993, Burge et al. 1999.Cis-splicing occurs in a two-step mechanism, each step consisting of a transesterification reaction (Moore et al. 1993) (Fig. 1A). The spliceosome, a ribonucleoprotein machinery composed of five small ribonucleoproteins (U1, U2, U4, U5, U6 snRNPs, named in relation to the kind of associated RNA molecule) and approximately 300 distinct proteins, is responsible for the splicing catalysis (Burge et al. 1999).Much effort was expended on the comprehension of the structure and dynamics of this complex machine during the splicing process, but the rules that discriminate introns and exons within the message are still poorly understood. Nevertheless, four conserved pre-mRNA sequence elements, which interact with the spliceosome, have been characterized as determinants in the splicing process (Moore et al. 1993, Burge et al. 1999 (Fig. 1C). In mammals, the 5' splice site consensus is AG/GURAGU (R for purine, /for splice site, bold for the dinucleotide 5' intron boundary) while the 3' splice site consensus is YAG/G (Y for pyrimidine, /for splice site, bold for the dinucleotides 3' intron boundary). There is also a conservation of nucleotide sequence around the branch point, CURAY (Y for pyrimidine, R for purine and A for branch Recently, other cis-regulatory elements were implicated in splice site recognition, e.g. the exonic splicing enhancers (ESEs) (Fig. 1C). ESEs are localized within exons and interact with a family of proteins rich in serine and arginine (SR proteins) that recruits the spliceosome to the proximal splice site .Although the presence of those consensuses was observed, it i...

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3′ splice site recognition in nematode trans-splicing involves enhancer- dependent recruitment of U2 snRNP

Cited by 15 publications

References 27 publications

Dual Function for U2AF³⁵ in AG-Dependent Pre-mRNA Splicing

Dual Function for U2AF³⁵ in AG-Dependent Pre-mRNA Splicing

Multiple Roles for SR Proteins intransSplicing

Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

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3′ splice site recognition in nematode trans-splicing involves enhancer- dependent recruitment of U2 snRNP

Cited by 15 publications

References 27 publications

Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing

Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing

Multiple Roles for SR Proteins intransSplicing

Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

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Dual Function for U2AF³⁵ in AG-Dependent Pre-mRNA Splicing

Dual Function for U2AF³⁵ in AG-Dependent Pre-mRNA Splicing