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Chapter 13. RNA Splicing. 生科二班 张瑾 学号： 200331060140. OUTLINE. Introduction The chemistry of RNA Splicing The Splicing Machinery Splicing Pathway Alternative Splicing Exon Shuffling RNA Editing mRNA Transport. Introduction.
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Chapter 13 RNA Splicing 生科二班 张瑾 学号：200331060140
OUTLINE • Introduction • The chemistry of RNA Splicing • The Splicing Machinery • Splicing Pathway • Alternative Splicing • Exon Shuffling • RNA Editing • mRNA Transport
Introduction • In almost all bacterial and phage genes,the opening-reading frame is a single stretch of condons with no break. • But the coding sequence of many eukaryotic genes is split into streches of condons interrupted by streches of noncoding sequence.
The coding streches in these split genes are called exons (for“expressed sequences”) The uncoding streches are called introns (for “intervening sequences”)
Note that: (1)The number of introns found within a gene varies enormously. (2)The site of the exons and introns vary.
The primary transcripts(pre-mRNA) must have their introns removed before they can be translated into protein. Introns are removed from the pre-mRNA by a process called RNA Splicing. RNA Splicing converts the pre-mRNA into mature messanger RNA.
The chemistry of RNA Splicing Sequence within the RNA determine where splicing occurs. • Specific nucleotide sequences within the pre-mRNA: • 5` splicing site:the exon-intron boundary at 5`end of the intron.(GU) • 3` splicing site:the exon-intron boundary at 3`end of the intron.(AG) • Branch point site:within the intron and is followed by Py tract.(A)
The intron is removed in a form called a lariat. Two transesterification reactions: 1)The 2`OH of the conserved A at the branch site attack the phosphoryl of the conserved G in the 5`splice site 2)The newly liberated 3`OH of the 5`exon attack the phosphoryl group at the 3`splice site. The newly liberated intron has the shape of a lariat.
Note that: 1)the chemistry of the process demand no energy input as it is just a question of shuffling bonds. 2)two features contribute to the direction of the splicing reaction: 1.An increase in entropy; 2.The excise intron is rapidly degraded.
In some cases, exons from different RNA molecules can be fused by Trans-Splicing. The Difference: A Y-shaped branch structure is formed instead of a lariat.
The splicing machinery RNA Splicing is carried out by a large complex called the spliceosome. Components involved: snRNPs; proteins not part of the snRNPs; proteins loosely bound to the spliceosome. It is believed that the RNA components rather than the proteins carry out the functions of the spliceosome.
snRNPs (small nuclear ribonuclear proteins) snRNP is composed of snRNAs and several proteins. The five snRNAs: U1,U2,U4,U5,U6 The snRNPs have 3 roles in splicing: • They recognise the 5`splice site and the branch site • They bring these sites together as required • They catalyze the RNA cleavage and joinging reactions.
Other proteins U2AF: Recognizes the Py tract/3`splice site and help BBP bind to the branch site. BBP RNA-annealing facters DEAD-box helicase proteins and ------
Splicing Pathway The splicing Pathway involves assembly,rearrangements and catalysis within the spliceosome. The action of the spliceosome is particularly interesting in two regards: First,the RNA components have a central role in recognizing introns and catalyzing their removal. Second,the complex is very dynamic.
Early complex Py tract A complex A is extruded and unpaired Tri-snRNP particle B complex U6 replace U1 U6 interacts with U2 C complex producing the active sit
The active site of the spliceosome is only formed on TNA sequences that pass the test of being recognized by multiple elements during spliceosome assembly. Yet,the problem of appropriate splice-site recognition in the pre-mRNA remains formidable: The splice sites are defined by rather short sequences with low levels of conservation.It thus represents a significant challenge for the splicing machinery to recognize and splice only at correct sites.
Splice-site recognition is prone to two kinds of errors: First, splice sites can be skipped; Second,other sites,close n sequence but not legitimate splice site,could be mistakenly recognized.
Two ways in which the accuracy of splice-site selection can be enhanced : First,it assembles on the sites soon after they have been synthesized. Second,there are other proteins---SR proteins---that bind near legitimate splice sites and help recruit the splicing machinery to those sites. So-called SR proteins bind to sequences called exonic splicing enhancers(ESEs)
Self-splicing Introns Apart from the pre-mRNA splicingmediated by Spliceosome found in eukaryotes,there are groupⅠand groupⅡ introns. They can remove themselves from RNAs in the tube in the absence of any proteins or other RNA molecules. But they are not enzymes,because they mediate only one round of RNA prodessing.
GroupⅡIntrons The chemistry of splicing ,and the RNA intermediates produced,are the same as for nuclear pre-mRNAs
GroupⅠIntrons GroupⅠ Introns release a linear rather a lariat. They use a free G nucleotide or nucleoside instead of a branch site A residue.
GroupⅠ Introns share a conserved secondary structure: • A binding pocket that will accommodate any guanine nucleotide or nucleoside. • An “internal fuide sequence”
Self-and spliceosome-mediated splicing The similar chemistry seen in self-and spliceosome-mediated splicing is believed to reflect an evolutionary relationship:perhaps ancestral groupⅡ-like self-splicing introns were the starting point for the evolution of modern pre-mRNA splicing.
RNA might carry out catalytic functions before! The structure of the catalytic region that performs the first transesterification reaction is very similar in the group Ⅱ introns and the pre-mRNA/snRNA complex.So, RNA might carry out catalytic functions before!
Alternative Splicing Single genes can produce multiple prodcts by alternative spling. Exons can be extended ,or skipped,also,introns can bbe retained in some messages.
Since there are mechanisms that ensure variations of this sort do not take place,how does alternative splicing occur so often? The basic answer is that some splice sites are used only some of the time ,leading to the production of different versions of the RNA from different transcriptions of the same gene. Constitutive alternative splicing:more than one product is always made from the transcribed gene. Regulated alternative splicing:different forms are generated at different conditions.
Examples: 1)Alternative splicing in the troponin T gene. 2)Constitutive alternative splicing.
Alternative spling is regulated by activators and repressors: Proteins regulating splicing bind to specific sites called : Exonic(or intronic) splicing enhancers(ESE or ISE) Exonic(or intronic) splicing silencers(ESS or ISS) The presence or activity of a given SR protein can determine whether a particular splice site is used in a particular cell type,or, at a particular stage of development,using its RS domain. An Example:the Drosophila Half-pint protein
Most silencers are recognized by members of the heterogeneous nuclear ribonucleoprotein(hnRNP) family which bind RNA but lack the RS domain ,by blocking specific splice sites.(acting as repressors) Examples: 1)hnRNPA1 2)the hnRNPI proein
A small group of introns are spliced by an alternative spliceosome compose of a different set of snRNPs In some organisms,certain pre-mRNAs are spliced by a low-abundance form of spliceosome----the AT-AC spliceosome. This rare form contains some components common to the major spliceosome but other unique components too. These unique components recognise different branch site and splice site squences ,but they both use the same chemical pathway.
A small group of introns are spliced by an alternative spliceosome compose of a different set of snRNPs In some organisms,certain pre-mRNAs are spliced by a low-abundance form of spliceosome----the AT-AC spliceosome. This rare form contains some components common to the major spliceosome but other unique components too. These unique components recognise different branch site and splice site squences ,but they both use the same chemical pathway.
It is suggested that the AT-AC introns evolved from the group Ⅱ introns and gives rise to the major pre-mRNA introns.
It is suggested that the AT-AC introns evolved from the group Ⅱ introns and gives rise to the major pre-mRNA introns.
Exon Shuffling Having the coding sequence of genes divided into several exons allows new genes to be be created by reshuffling exons. The borders between exons and introns within a given gene often coincide with the boundaries between domains within the protein encoded by that gene. • Many genes,and the proteins they encode,have apparently arisen during evolution in part via exon dupllication and divergence.
Related genes are sometimes found in otherwise unrelated genes. An example :LDL receptor gene We can see from all above that it has been relatively easy ,through evolution,to generate new proteins by shuffling existing exons between genes.
RNA Editing RNA Editing is another mechanism that allows an RNA to be changed after transcription so as to encode a different protein from that encoded by the gene. • There are two mechanisms that mediate editing: • Site-specific deamination • Guide RNA-directed uridine insertion or deletion
Site-specific deamination In one form of site-specific deamination,a specifically targeted cyosine residue within mRNA is converted into uridine by deamination. An Example : Mammalian apolipoprotein-B gene
Other forms of deaminations include adenosine deamination which is carried out by the enzyme ADAR(adenosine deaminase acting on RNA). ADAR adenosine Inosine
Guide RNA-directed uridine insertion or deletion direct gRNA to the region it will edit determine where Us are inserted Poly-U streches
mRNA transport RNA export from the nucleus is an active process. Only certain RNAs are selected for transport. The RNA has the correct collection of proteins bound to it: Protein that recognise exon:exon boundaries,indicating an mature mRNA appropriately spliced:such as SR protein. Protein that bind introns indicate an RNA to be retained .
Export takes placethrough a special structure in the nuclear membrame calle the nuclear pore complex. Small molecular: unaided Larger molecular: require active transport which is supplied by hydrolysis of GTP
