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12.6: Splicing of introns in pre‑mRNAs - Biology

12.6: Splicing of introns in pre‑mRNAs - Biology


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1. Splice Sites

The sequence at the 5' and 3' ends of introns in pre-mRNAs is very highly conserved. Thus one can derive a consensus sequence for splice junctions.

5' exon...AG'GURAGU.................YYYYYYYYYYNCAG'G....exon

The GU is the 5' splice site (sometimes called the donor splice site) and the AG is the 3' splice site (or acceptor splice site). GU is invariant at the 5' splice site, and AG is (almost) invariant at the 3' splice site for the most prevalent class of introns in pre-mRNA.

Effects of mutations at the splice junctions demonstrate their importance in the splicing mechanism. Mutation of the GT at the donor site in DNA to an AT prevents splicing (this was seen in a mutation of the b‑globin gene that caused b0 thalassemia.) A different mutation of the b‑globin gene that generated a new splice site caused an aberrant RNA to be made, resulting in low levels of b‑globin being produced (b+ thalassemia).

2. The intron is excised as a lariat

The 2'‑OH of an A at the "branch" point forms a phosphoester with the first G of the intron to initiate splicing. Splicing occurs by a series of phosphoester transfers (also called trans‑esterifications). After the 2'-OH of the A at the branch has joined to the initial G of the intron, the 3'‑OH of the upstream exon is available to react with the first nucleotide of the downstream exon, thereby joining the two exons via the phosphoester transfer mechanism.

c. Intron lariat is the equivalent of a "circular" intermediate.

The sequence at the branch point is only moderately conserved in most species; examination of many branch points gives the consensus YNYYRAG. It lies 18 to 40 nucleotides upstream of the 3' splice site.

3. Small nuclear ribonucleoproteins (or snRNPs) form the functional splicesome on pre‑mRNA and catalyze splicing.

a. "U" RNAs and associated proteins. Small nuclear RNAs (snRNAs) are about 100 to 300 nts long and can be as abundant as 105 to 106 molecules per cell. They are named U followed by an integer. The major ones involved in splicing are U1, U2, U4/U6, and U5 snRNAs. They are conserved from yeast to human. The snRNAs are associated with proteins to form small nuclear ribonucleoprotein particles, or snRNPs. The snRNPs are named for the snRNAs they contain, hence the major ones involved in splicing are U1, U2, U4/U6, U5 snRNPs.

One class of proteins common to many snRNPs are the Sm proteins. There are 7 Sm proteins, called B/B’, D1, D2, D3, E, F, G. Each Sm protein has similar 3-D structure, consisting of an alpha helix followed by 5 beta strands. The Sm proteins interact via the beta strands, and may form circle around RNA.

A particular sequence common to many snRNAs is recognized by the Sm proteins, and is called the “Sm RNA motif”.

b. Use of antibodies from patients with SLE. Several of the common snRNPs are recognized by the autoimmune serum called anti‑Sm, initially generated by patients with the autoimmune disease Systemic Lupus Erythematosis. One of the critical early experiments showing the importance of snRNPs in splicing was the demonstration that anti-Sm antisera is a potent inhibitor of in vitrosplicing reactions. Thus the targets of the antisera, i.e. Sm proteins in snRNPs, are needed for splicing.

c. The snRNPs assemble onto the pre-mRNA to make a large protein-RNA complex called a spliceosome (Figure 3.3.17). Catalysis of splicing occurs within the spliceosome. Recent studies support the hypothesis that the snRNA components of the spliceosome actually catalyze splicing, providing another example of ribozymes.

d. U1 snRNP: Binds to the 5' splice site, and U1 RNA forms a base‑paired structure with the 5' splice site.

e. U2 snRNP: Binds to the branch point and forms a short RNA-RNA duplex. This step requires an auxiliary factor (U2AF) and ATP hydrolysis, and commits the pre-mRNA to the splicing pathway.

f. U5 snRNP plus the U4, U6 snRNP now bind to assemble the functional spliceosome. Evidence indicates that U4 snRNP dissociates from the U6 snRNP in the spliceosome. This then allows U6 RNA to form new base-paired structures with the U2 RNA and the pre-mRNA that catalyze the transesterification reaction (phosphoester transfers). One model is that U6 RNA pairs with the 5' splice site and with U2 RNA (which itself is paired to the branch point), thus bringing the branch point A close to the 5' splice site. U5 RNA may serve to hold close together the ends of the exons to be joined.

4. Trans‑splicing

All of the splicing we have discussed so far is between exons on the same RNA molecule, but in some cases exons can be spliced to other RNAs. This is very common in trypanosomes, in which a spliced leader sequence is found at the 5' ends of almost all mRNAs. A few examples of transsplicing have been described in mammalian cells.


Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.


Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses

Precursor mRNAs with introns can undergo alternative splicing (AS) to produce structurally and functionally different proteins from the same gene. Here, we show that the pre-mRNAs of Arabidopsis genes that encode serine/arginine-rich (SR) proteins, a conserved family of splicing regulators in eukaryotes, are extensively alternatively spliced. Remarkably about 95 transcripts are produced from only 15 genes, thereby increasing the complexity of the SR gene family transcriptome by six-fold. The AS of some SR genes is controlled in a developmental and tissue-specific manner. Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) dramatically altered the AS of pre-mRNAs of several SR genes, whereas hormones altered the splicing of only three SR genes. These results indicate that abiotic stresses regulate the AS of the pre-mRNAs of SR genes to produce different isoforms of SR proteins that are likely to have altered function(s) in pre-mRNA splicing. Sequence analysis of splice variants revealed that predicted proteins from a majority of these variants either lack one or more modular domains or contain truncated domains. Because of the modular nature of the various domains in SR proteins, the proteins produced from splice variants are likely to have distinct functions. Together our results indicate that Arabidopsis SR genes generate surprisingly large transcriptome complexity, which is altered by stresses and hormones.

Figure S1. Amplification of Arabidopsis SR genes by PCR. Arabidopsis genomic DNA (A) or DNAse-treated RNA (B) was amplified with gene-specific primers of SR genes. The primer sets used for amplification are indicated in Table S1 and Figure 4. The amplified products were separated on an agarose gel and stained with ethidium bromide. The name of the SR gene is indicated on top of each lane. Lane M, DNA size markers. Figure S2. Expression of SR genes in different organs and pollen. Each transcript was quantified and normalized to cyclophilin. The total of all isoforms in each organ/pollen for each gene is presented. Figure S3. Expression of SR genes in 3-, 5-, 10- and 15-day-old seedlings. Each transcript was quantified and normalized to cyclophilin. The total of all isoforms at each stage for each gene is presented. Figure S4. Verification of the effectiveness of various stress and hormonal treatments using genes that are known to be induced by these signals. A) Induction of known genes by various treatments. Two-week-old seedlings were subjected to various treatments as described in the legend of Figure 3. RNA from the control and treated samples was used for RT-PCR analyses. The presence of an equal quantity of template in each reaction was verified by amplifying the cyclophilin transcript (bottom panel in Figure 3a and Figure S6). RD29A, a gene induced by ABA, NaCl and cold IAA1, an IAA-induced gene ARR4, cytokinin-induced response regulator HSP18, heat-shock protein 18, a heat-induced gene HXK1, hexokinase 1, a glucose-induced gene PR1, pathogenesis-related 1. B) Quantification of induction of positive control genes in response to hormones and stress. The level of transcript was quantified and normalized to cyclophilin (see Figure 3a and Figure S6). Figure S5. Expression of SR genes in seedlings that are subjected to various treatments. Each isoform was quantified and normalized to cyclophilin. The total of all isoforms in each treatment for each gene is presented. Figure S6. Effect of hydrogen peroxide and methyl jasmonate on the AS of SR genes. Seedlings were treated with either 100 μM hydrogen peroxide (H202) for 6 h or 100 μM methyl jasmonate (MJ) for 6 h as described in Experimental procedures. Figure S7. The effect of glucose and mannitol on alternative splicing. A) Gel pictures showing the alternative splicing pattern. B) Quantification of splice variants. Each splice variant was quantified and normalized to cyclophilin and presented as the percentage of all isoforms in a sample. Table S1. Sequences of gene-specific primers of Arabidopsis SR genes used in RT-PCR. Table S2. Sequences of gene-specific primers of positive control genes.

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Alternative Splicing of Pre-Messenger RNAs in Plants in the Genomic Era

AbstractPrimary transcripts (precursor-mRNAs) with introns can undergo alternative splicing to produce multiple transcripts from a single gene by differential use of splice sites, thereby increasing the transcriptome and proteome complexity within and between cells and tissues. Alternative splicing in plants is largely an unexplored area of gene expression, as this phenomenon used to be considered rare. However, recent genome-wide computational analyses have revealed that alternative splicing in flowering plants is far more prevalent than previously thought. Interestingly, pre-mRNAs of many spliceosomal proteins, especially serine/arginine-rich (SR) proteins, are extensively alternatively spliced. Furthermore, stresses have a dramatic effect on alternative splicing of pre-mRNAs including those that encode many spliceosomal proteins. Although the mechanisms that regulate alternative splicing in plants are largely unknown, several reports strongly suggest a key role for SR proteins in spliceosome assembly and regulated splicing. Recent studies suggest that alternative splicing in plants is an important posttranscriptional regulatory mechanism in modulating gene expression and eventually plant form and function.


The spatial distributions of pre-mRNAs suggest post-transcriptional splicing of specific introns within endogenous genes

Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes’ transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.


Role of the 3′ Splice Site in U12-Dependent Intron Splicing

Fig. 1 . Distribution of branch site-to-3′ splice site distances in putative U12-dependent introns. The data are from the compilation of putative U12-dependent introns in Burge et al. (5). The solid line shows the distribution for all introns, while the black and grey bars show the distribution of distances in the AU-AC and GU-AG subclasses of U12-dependent introns, respectively. The branch site is assumed to be the position of the second adenosine in the branch site consensus sequence UCCUUAAC. All branch sites could be aligned with the consensus without ambiguity. In a few cases, the presumed branch site was a G residue.

Nucleotide requirements for 3′ splice site function in vivo.

Fig. 2 . Sequences of the 3′ splice site mutants used in this study. The common 5′ splice site is shown at the left. The 3′ splice sites are shown at the right. The common branch site is shown in bold, and the mutated positions are underlined. The sites of in vivo splicing for each construct are indicated by the arrows. Major sites are indicated by dark arrowheads, and minor sites are indicated by light arrowheads. The numbers at the top refer to the distance in nucleotides between the branch site adenosine and the dashed lines. Fig. 3 . Pattern of in vivo splicing of 3′ splice site dinucleotide mutants. The indicated minigene constructs were transfected into CHO cells, and total RNA was prepared after 48 h. The splicing pattern of the P120 intron F was determined by RT-PCR amplification and seperation of the products on a denaturing gel. The positions of spliced and unspliced PCR products are shown, as well as the position of a U2-dependent cryptic splice that is activated in some constructs. The numbers at the left indicate the distance in nucleotides between the branch site and the 3′ splice site in each product.

Spacing requirements for 3′ splice site function in vivo.

Fig. 4 . Pattern of in vivo splicing of 3′ splice site spacing mutants. The analysis and presentation are as in Fig. 3.

Spacing mutants do not activate cryptic branch sites.

Fig. 5 . Cryptic U12-dependent 3′ splice site usage in branch and 3′ splice site mutants. The indicated minigene constructs were transfected and assayed as in Fig. 3 except that a primer at the 3′ end of exon 7 was used in the PCR amplification and the products were seperated on an agarose gel. Lane 2 shows the activation of the cryptic U12-dependent 3′ splice site at position 124 of exon 7 when the branch site is mutated from the consensus sequence UCUUAAC to AGUUAAC. Lane 10 is 50-bp ladder molecular size markers (Life Technologies).

In vitro effects of alterations of the 3′ splice site.

Fig. 6 . In vitro splicing patterns of 3′ splice site constructs. Templates for in vitro transcription of the indicated 3′ splice site constructs were produced by PCR amplification from the minigene constructs tested in vivo. Equal amounts of transcribed precursor RNAs were spliced in vitro. All reactions contained an anti-U2 snRNA 2′-O-methyl oligonucleotide which inhibits U2-dependent splicing. An anti-U12 snRNA 2′-O-methyl oligonucleotide was also added to even-numbered lanes to inhibit U12-dependent splicing. The structures of the various RNA products are shown on the left and correspond, from top to bottom, to the unspliced precursor, spliced exon product, the exon 1 intermediate generated by the first step of splicing, and the excised lariat intron generated by the second step of splicing. The lariat introns from different constructs migrate differently due to the varying length of RNA 3′ of the branch. A degradation product which migrates below the position of spliced exons is labeled with an asterisk. The splicing reactions shown in lanes 17 and 18 used a truncated precursor RNA that terminated 7 nucleotides 3′ of the branch site and thus was missing the 3′ splice site and the downstream exon. Fig. 7 . RT-PCR analysis of the products of in vitro splicing. RNA was isolated from the position of spliced exons in a preparative version of the experiment shown in Fig. 6, reverse transcribed using a primer near the 3′ end of the precursor, and PCR amplified using a nested 3′ primer and a 5′ primer in the first exon, followed by denaturing gel electrophoresis to determine the sites of splicing. Due to differences in RNA recovery and amplification, differences in band intensities between lanes are not quantitatively meaningful. Fig. 8 . Spliceosome formation activity of 3′ splice site constructs. Equal amounts of the in vitro-transcribed RNA used in Fig.6 were incubated under U12-dependent splicing conditions for the times indicated, treated with heparin, and separated on nondenaturing gels. The splicing-specific complexes A and B are well resolved from the nonspecific complex H. Lane 1 shows complexes formed on a U2-dependent adenovirus major late intron precursor incubated in the absence of the anti-U2 snRNA oligonucleotide.

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Lariat Structure. Lariat chain, a science demonstration. Splicing of group ii introns.

A type of long necklace (= a piece of…. Structures of a binuclear catalytic mutant in. He surmises that the lariat structure had escaped detection for so long because of its transient nature. So what does the lariat structure teach us about splicing by the spliceosome? A professional wrestling attack, a move.

RNA splicing - the lariat conformation captured at PROXIMA . from www.synchrotron-soleil.fr Structure of a split yeast gene: (a) working principle of the dilator. Thinking that every biological structure should have a purpose usually comes from a confusion between the general evolution process and what is. Also, to lasso or catch with a lariat. He surmises that the lariat structure had escaped detection for so long because of its transient nature.

Lariat structure in anderen sprachen:

A type of long necklace (= a piece of…. The lariat conformation helps assemble the group ii active site for the reverse splicing reaction. Browse the use examples 'lariate structure' in the great english corpus. Lariat — lar i*at (la^r i^*a^t), v. Click on the first link on a line below to go directly to a page where lariat structure is defined. A branched (lariat) structure is formed during. Complete nucleotide sequence of the actin gene in saccharomyces cerevisiae. A professional wrestling attack, a move. Lariat structure in anderen sprachen: A rope necklace long enough to loop several times around the neck. Splicing of group ii introns. Lariat rnas as intermediates and products in the splicing of messenger rna precursors. We found one dictionary with english definitions that includes the word lariat structure:

Lariat may also refer to: A type of long necklace (= a piece of…. A rope used for catching or tying up an animal: Determined the structure of an excised group ii intron in its branched conformation. Figure 14.7 defining the branched lariat in splicing.

Molecular characterization of a new member of the lariat . from media.springernature.com A professional wrestling attack, a move. Click on the first link on a line below to go directly to a page where lariat structure is defined. A genetic structure in rna splicing. A lariat is a rope in the form of a lasso. A rope used for catching or tying up an animal:

Figure 14 7 defining the branched lariat in splicing.

We found one dictionary with english definitions that includes the word lariat structure: Both steps involve reactions that occur between rna nucleotides. Figure 14 7 defining the branched lariat in splicing. A rope used for catching or tying up an animal: A lariat is a rope in the form of a lasso. Also, to lasso or catch with a lariat. This is the group that most thing of when speaking a complex of proteins and snrnas (the spliceasome) is required. Lariat structure in anderen sprachen: Lariat rnas as intermediates and products in the splicing of messenger rna precursors. .] to secure with a lariat fastened to a stake, as a horse or mule for grazing Structure of a split yeast gene: Figure 1 the working principle and structure of the mechanical dilatation device: This lariat like structure acts as an intermediate.

Figure 14.7 defining the branched lariat in splicing. Here, we report the first structures of dbr1 alone and in complex with several synthetic rna compounds that mimic the branchpoint in lariat rna. A branched (lariat) structure is formed during. Lariat structure in anderen sprachen: Determined the structure of an excised group ii intron in its branched conformation.

12.6: Splicing of introns in pre‑mRNAs - Biology LibreTexts from www.personal.psu.edu This is the group that most thing of when speaking a complex of proteins and snrnas (the spliceasome) is required. Also, to lasso or catch with a lariat. Splicing of group ii introns. Structure of a split yeast gene: Lariat rnas as intermediates and products in the splicing of messenger rna precursors.

He surmises that the lariat structure had escaped detection for so long because of its transient nature.

A type of long necklace (= a piece of…. The lariat conformation helps assemble the group ii active site for the reverse splicing reaction. Complete nucleotide sequence of the actin gene in saccharomyces cerevisiae. Check out the pronunciation, synonyms and grammar. Lariat structure in anderen sprachen: Determined the structure of an excised group ii intron in its branched conformation. This lariat like structure acts as an intermediate. A lariat is a rope in the form of a lasso. Learn the definition of 'lariate structure'. A genetic structure in rna splicing. Splicing of group ii introns. He surmises that the lariat structure had escaped detection for so long because of its transient nature. The structure of the lariat was very provocative, menees said.

Source: www.researchgate.net

Check out the pronunciation, synonyms and grammar. Complete nucleotide sequence of the actin gene in saccharomyces cerevisiae. .] to secure with a lariat fastened to a stake, as a horse or mule for grazing A branched (lariat) structure is formed during. Both steps involve reactions that occur between rna nucleotides.

Source: www.researchgate.net

Browse the use examples 'lariate structure' in the great english corpus. A genetic structure in rna splicing. Thinking that every biological structure should have a purpose usually comes from a confusion between the general evolution process and what is. Structure of a split yeast gene: Structures of a binuclear catalytic mutant in.

A branched (lariat) structure is formed during. This lariat like structure acts as an intermediate. Lariat may also refer to: Also, to lasso or catch with a lariat. Figure 14.7 defining the branched lariat in splicing.

Source: febs.onlinelibrary.wiley.com

Learn the definition of 'lariate structure'. We found one dictionary with english definitions that includes the word lariat structure: A genetic structure in rna splicing. So what does the lariat structure teach us about splicing by the spliceosome? Thinking that every biological structure should have a purpose usually comes from a confusion between the general evolution process and what is.

Source: www.researchgate.net

Lariat structure in anderen sprachen: This lariat like structure acts as an intermediate. A lariat is a rope in the form of a lasso. Browse the use examples 'lariate structure' in the great english corpus. Figure 14.7 defining the branched lariat in splicing.

Source: www.researchgate.net

.] to secure with a lariat fastened to a stake, as a horse or mule for grazing

Complete nucleotide sequence of the actin gene in saccharomyces cerevisiae.

Source: www.researchgate.net

(a) working principle of the dilator.

So what does the lariat structure teach us about splicing by the spliceosome?

Source: live.staticflickr.com


Group II intron splicing factors in plant mitochondria

Group II introns are large catalytic RNAs (ribozymes) which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA) in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has been retained in the mtDNA of various angiosperms: the matR gene encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4). Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4), which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory splicing cofactors.

Keywords: group II intron maturase mitochondria plant respiration splicing splicing factor.


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