Rho independent termination
Scheme 3: Splice sites in most of the vertebrates
S. No. snRNPs Size (nucleotides) Role
1 U1 snRNP 165 Binds to 5’ splice site and then the 3’ splice site;
Promotes binding of U2 snRNP
2 U2 snRNP 185 Binds the branch site (aided by U2AF);
Forms part of catalytic center after binding with U6
3 U5 snRNP 116 Binds 5’ splice site (A loop in U5 snRNA is immediately adjacent to the first base positions in both exons)
4 U4 snRNP 145 Masks the catalytic activity of U6 5 U6 snRNP 106 Catalyzes splicing
Splicing of nuclear pre-mRNA consists of two successive transesterification reactions, in which phosphodiester bonds within the pre-mRNA are broken and new ones are formed. Thus, the number of phosphodiester bonds remains constant during reactions. The chemistry of the splicing process is simple. The intron is removed in a form called a lariat as the flanking exons are joined (Fig. 23).
Thus, the snRNPs have three roles in splicing reaction:
& To recognize 5’ splice site and branch site and bring them together as required
& To catalyze (or help to catalyze) the RNA cleavage and joining reactions Various steps of splicing pathway can be summarized as follows:
$ Formation of Early (E) complex: Initially, the 5’ splice site is recognized by the U1 snRNP (using base pairing between its snRNA and the pre-mRNA). One subunit of U2 auxillary factor (U2AF) binds to the pyrimidine tract and the other to the 3’ splice site. The former subunit interacts with branch point binding protein (BBP) and helps that protein to bind to the branch site. This arrangement of proteins and RNA is called the Early (E) complex.
$ Formation of A complex: U2 snRNP then binds to the branch site, aided by U2AF and displacing BBP. This arrangement is called the A complex. The base pairing between the U2 snRNA and the branch site is such that the branch site ‘A’ residue is extruded from the resulting stretch of double helical RNA as a single nucleotide bulge. This ‘A’ residue is thus unpaired and available to react with the 5’ splice site.
5'
A G
G U A A G U
Y R Y N Y 2'OH
P 5' splice site
Exon 1
Intron
Precursor mRNA Yn N C
A A G 3'
G 3' splice site P
Exon 2
Branch site
5' A G
G U A A G U
Y R Y N Y P
Intron Yn N C
A A G 3'
G P
2'
5'
3' 3'OH
Lariat intermediate First
transesterification reaction
Second transesterification reaction
G U A A G U
Y R Y N Y P
Intron 2'
5'
3'
Lariat form of intron Yn N C 3'OH
A A G
5' A G
Exon 1 3'
G P Exon 2
+
Spliced product
Fig. 23: Splicing of nuclear pre-mRNA
$ Formation of B complex: The next step is a rearrangement of the A complex to bring together all three splice sites. This is achieved as follows: the U4 snRNP and U6 snRNP, along with the U5 snRNP, join the complex. Together these three snRNPs are called tri-snRNP particle, within which the U4 snRNP and U6 snRNP are held together by complementary base pairing between their RNA components and the U5 snRNP is more loosely associated through protein-protein interactions. With the entry of the tri-snRNP, the A complex is converted into the B complex.
$ Formation of C complex: In the next step, U1 snRNP leaves the complex and U6 snRNP replaces it at the 5’ splice site. This requires that the base pairing between the U1 snRNA and the pre-mRNA be broken, allowing the U6 snRNA to anneal with the same region (infact, to an overlapping sequence). These steps complete the assembly pathway. The next rearrangement triggers catalysis and occurs as follows: U4 snRNP is released from the complex, allowing U6 snRNP to interact with U2 snRNP (through RNA:RNA base pairing). This rearrangement is called the C complex. This rearrangement has following consequences:
& It produces the active site. It brings together those components believed to be solely regions of the U2 snRNA and U6 snRNA within the spliceosome forming the active site.
& The same rearrangement also ensures the proper positioning of the substrate RNA to be acted upon.
& Formation of the active site juxtaposes the 5’ splice site pre-mRNA and the branch site.
It is striking that the active site is primarily formed of RNA, but also that it is only formed at this
stage of spliceosome assembly. Presumably this strategy lessens the chance of aberrant splicing.
Linking the formation of the active site to the successful completion of the earlier steps in spliceosome assembly makes it highly likely that the active site is available only at legitimate splice sites.
$ Joining of exons and release of mature mRNA: The juxtaposition of the 5’ splice site pre-mRNA and the branch site facilitates the first transesterification reaction. The second reaction, between the 5’ and 3’ splice sites, is aided by the U5 snRNP, which helps to bring the two exons together. The final steps involve release of mRNA product and the snRNPs. The snRNPs are initially bound to the lariat, but get recycled after rapid degradation of that piece of RNA.
Components of the splicing machinery arrive or leave the complex at each step due to changes associated with structural rearrangements necessary for the splicing reaction to proceed. There is evidence to suggest that some of the components shown do not arrive or leave precisely when indicated in the figure, they may, for eg., remain present but weaken their association with the complex rather than dissociating completely. It is also not possible to be sure of the order of some changes shown, particularly the two steps involving changes in U6 pairing: when it takes over from U1 snRNP at the 5’ splice site, compared to when it takes over from U4 snRNP in binding U2 snRNP. Despite these uncertainties, the critical involvement of different components of the machinery at different stages of the splicing reaction and the general dynamic nature of the spliceosome, are as shown in Fig. 24.
Some eukaryotic pre-mRNAs do not fall into the GU-AG intron category. They have different consensus sequences at their splice sites. These are AU-AC introns, which have been found in approximately 20 genes in organisms as diverse as humans, plants and Drosophila. These introns require U11 / U12 snRNPs.
(c) Polyadenylation of 3’ end (followed by termination of transcription): The final RNA processing event, polyadenylation of the 3’ end of pre-mRNA, is intimately linked with the termination of transcription. Just as with capping and splicing, the polymerase CTD tail is involved in recruiting the enzymes necessary for polyadenylation. Once polymerase has reached the end of a gene, it encounters specific sequences that, after being transcribed into RNA, trigger the transfer of polyadenylation enzymes to that RNA, leading to three events (Fig. 25):
& Cleavage of the message
& Addition of many adenine residues to its 3’ end by Poly A polymerase
& Termination of transcription by polymerase
Eukaryotic mature mRNA transcripts have more nucleotides beyond 3’ end. Indeed, the nucleotide preceding the poly (A) is not the last nucleotide to be transcribed.
Polyadenylation was once looked on as a ‘post transcriptional’ event but it is now recognized that the process is an inherent part of the mechanism for termination of transcription by RNA Pol II.
5' A 3'
A BBP
U2 snRNP
5' A 3'
BBP U2AF65 35 U1 snRNP
U6 snRNP
U4 snRNP
+
U5 snRNP
U2AF6535
A
U1 snRNP
A
A
U4 snRNP
A
Lariat form of intron Spliced exons
snRNP particle Tri
3' 5'
Spliceosomal mediated splicing reaction:
Fig. 24: Spliceosomal mediated slicing
In mammals, polyadenylation is directed by a signal sequence in the mRNA, almost invariably 5’-AAUAAA-3’. These are cleaved by a specific endonuclease that recognizes the sequence AAUAAA. Cleavage does not occur if this sequence or a segment of some 20 nucleotides on its 3’ side is deleted. The presence of internal AAUAAA sequences in some mature mRNAs indicates that AAUAAA is only part of the cleavage signal. This sequence is located between 10 and 30 nucleotides upstream of the dinucleotide 5’-CA-3’ and is followed 10-20 nucleotides later
by a GU rich region. Both the poly (A) signal sequence and the GU rich region are binding sites for multisubunit protein complexes.
& Cleavage and polyadenylation specificity factor (CPSF) binds poly (A) signal sequence.
& Cleavage stimulation factor (CstF) binds GU rich region.
5’ 3’
Cleavage site
Ongoing transcription
AAAAAAAAAAAAA
5’ Poly A
Pol
Fig. 25: Termination step involves cleavage followed by polyadenylation of transcript Besides, Poly (A) polymerase and at least two other protein factors must associate with bound CPSF and CstF in order for polyadenylation to occur.
After cleavage by the endonuclease, template-independent RNA polymerase called poly (A) polymerase adds about 250 adenylate residues to the 3’ end of the transcript. Virtually, all eukaryotic mRNAs have a series of up to 250 adenosines at their 3’ ends. This enzyme uses ATP as a precursor and adds ‘A’ residues using the same chemistry as RNA polymerase. These ‘A’
residues are not specified by DNA sequence, i.e. these A(s) are added without a template. Thus, the long tail of A(s) is found in the RNA but not the DNA. It is not clear what determines the length of the poly A tail, but that process involves other proteins that bind specifically to the poly A sequence (described later). The polymerase does not act at the extreme 3’ end of the transcript, but at an internal site, which is cleaved to create a new 3’ end to which the poly (A) tail is added.
The reaction catalyzed is as follows:
RNA + n ATP → RNA-(AMP)n + PPi
The additional factors required include polyadenylate-binding protein (PABP). These PABPs catalyze the following functions:
& To help the polymerase to add the adenosines
& Possibly influences the length of the poly (A) tail that is synthesized
& Appears to play a role in maintenance of the tail after synthesis
& Also play a role in translation
In yeast, the signal sequences in the transcript are slightly different, but the protein complexes are similar to those in mammals and polyadenylation is thought to occur by more or less the
same mechanism.
CPSF is known to interact with TFIID and is recruited into the polymerase complex during the initiation stage. By riding along the template with RNA Pol II, CPSF is able to bind to the poly (A) signal sequence as soon as it is transcribed, initiating the polyadenylation reaction. Both CPSF and CstF contact with the CTD of the polymerase. It has been suggested that the nature of these contacts changes when the poly (A) signal sequence is located and that this change alters the properties of the elongation complex so that termination becomes favored over continued RNA synthesis. As a result, transcription stops soon after the poly (A) signal sequence has been transcribed. The details of the termination step linking cleavage and polyadenylation to termination of transcription are outlined in Fig. 26.
It is noteworthy that the long tail of A(s) is unique to transcripts made by RNA Pol II, a feature that allows experimental isolation of protein coding mRNAs by affinity chromatography. The mature mRNA is then transported from the nucleus.
It is not known what links polyadenylation to termination, but it is clear that the polyadenylation signal is required for termination (interestingly, RNA cleavage is not). Two basic models have been proposed to explain the link between polyadenylation and termination:
& First that the transfer of 3’ processing enzymes from the polymerase CTD tail to the RNA triggers a conformational change in the polymerase that reduces processivity of the enzyme, leading to spontaneous termination soon afterward.
& The second model proposes that the absence of a 5’ cap on the second RNA molecule is sensed by the polymerase, which, as a result, recognizes the transcript as improper and terminates. The absence of the cap reflects the absence of the capping enzymes on the CTD at this stage of the transcription cycle (these enzymes are loaded onto the CTD at the point where initiation turns to elongation and are then displaced in favor of the splicing machinery).
The role of poly (A) tail is still not firmly established despite much effort. Even though polyadenylation can be identified as an inherent part of the termination process, this does not explain the necessity to add a poly (A) tail to the transcript. Evidence that it enhances translation efficiency and the stability of mRNA is accumulating. The poly (A) tail on pre-mRNA is thought to help stabilize the molecule since a poly (A)-binding protein binds to it, which should act to resist 3’ exonuclease action. In addition, the poly (A) tail may help in the translation of the mature mRNA in the cytoplasm. Blocking the synthesis of poly (A) tail by exposure to 3’- deoxyadenosine (cordycepin) does not interfere with the synthesis of primary transcript. The mRNA devoid of a poly (A) tail can be transported out of the nucleus. However, an mRNA molecule devoid of a poly (A) tail is usually a much less effective template for protein synthesis than is one with a poly (A) tail. Thus, poly (A) tail has a role in initiation of translation. It is further supported by research showing that poly (A) polymerase is repressed during those periods of the cell cycle when relatively little protein synthesis occurs. Indeed some mRNAs are stored in an unadenylated form and receive the poly (A) tail only when translation is imminent.
The half-life of an mRNA molecule may also be determined in part by the rate of degradation of its poly (A) tail. Histone pre-mRNAs do not get polyadenylated, but are cleaved at a special sequence to generate their mature 3’ ends.
5’………..AAUAAA…………..CA….…GU rich region………3’
5’………..AAUAAA…………..CA…..…GU rich region………3’
5’………..AAUAAA…………..CAAAAAAAAAAAAAA3’
Polyadenylated mRNA Pre-mRNA
RNA Pol II
Polyadenylate binding protein
CstF 10-30 bp 10-20 bp
Polyadenylation CPSF
DNA
DNA Polyadenylation
signal sequence (AAUAAA)
CPS F RNA
RNA CstF
CstF
Termination is favored over
CPSF is shown attached to the RNA Pol II elongation complex that is synthesizing RNA. CPSF binds to the polyadenylation signal sequence AAUAAA as soon as it is transcribed. This changes the interaction between CPSF and the CTD of RNA Pol II so that termination of transcription is now favored over continued elongation. CstF probably attaches the GU rich region downstream of AAUAAA. The CPSF is shown to leave the complex in order to bind to the polyadenylation signal, when in reality it may maintain its attachment to RNA Pol II during the polyadenylation process.
Cleavage proteins attaches to signal sequence CPSF
Fig. 26: Termination signal and the link between polyadenylation and termination of transcription by RNA Pol II
(d) Pre-mRNA methylation: The final modification or processing event that many pre- mRNA undergo is specific methylation of certain bases. In vertebrates, the most common methylation event is on the N6 position of A residues, particularly when these A residues occur in the sequence 5’-RRACX-3’, where X is rarely G. Up to 0.1% of pre-mRNA A residues are methylated and the methylations seem to be largely conserved in the mature mRNA, though their function is unknown.
Alternative mRNA processing
Alternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.
Alternative processing can be achieved in four different ways:
& By using different poly (A) sites
& By using different promoters
& By retaining certain introns / by retaining or removing certain exons
& RNA editing
(a) Alternative poly (A) sites: Some pre-mRNAs contain more than one poly (A) site and these may be used under different circumstances (eg. in different cell types) to generate different mature mRNAs. The cell or organism has a choice of which one to use. It is possible that if the upstream site is used then sequences that control mRNA stability or location are removed in the portion that is cleaved off. Thus mature mRNAs with the same coding region, but differing stabilities or locations, could be used in the same cell at a frequency that reflects their relative efficiencies (strengths) and the cell would contain both types of mRNA. The efficiency of a poly (A) site may reflect how well it matches the consensus sequences. In other situations, one cell may exclusively use one poly (A) site, while a different cell uses another. The most likely explanation is that in one cell the stronger site is used by default, but in the other cell a factor is present that activates the weaker site so it is used exclusively, or that prevents the stronger site from being used. In some cases, the use of alternative poly (A) sites causes different patterns of splicing to occur. In some cases, factors will bind near to and activate or repress a particular site.
(b) Alternative promoters: The use of different promoters in different cell types and at different developmental stages lead to the generation of different mature mRNAs.
(c) Alternative splicing: In many cases, the generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’-splice sites. This is called alternative splicing. Hence, a single transcript can be spliced in multiple ways resulting in a number of protein coding sequences.
The alternative splicing events as depicted in Fig. 27 are:
& Exon skipped
& Intron retained
& Exon extended using cryptic splice sites
& Alternative exons
Intron 2
DNA
Exon 1 Exon 2 Exon 3 Intron 1 Intron 2
Primary transcript
5' 3'
5' 3'
RNA Transcription
Exon 1Exon 2Exon 3Exon 1Exon 3Exon 1Exon 2Exon 3 Exon 1Intron 1Exon 2Exon 3
Exon 2 Exon 1
+ Exon 3 Exon 1
Spliced mRNA Splicing
Normal Exon skipped Exon extended Intron retained Alternative exons
Exon on xon 3
Intron 1
1 Ex 2 E
Fig. 27: Types of splicing
By this strategy, a gene can give rise to more than one polypeptide product with partially
As these splicing events occur differently in different cell types, it is likely that cell type-specific
d) RNA editing: An unusual form of RNA processing in which the sequence of the primary
There are two major mechanisms that mediate editing:
ich a substitution overlapping sequences and is more common in higher eukaryotes. Some pre-mRNAs can be spliced in more than one way, generating alternative mRNAs. It is estimated that 30% of the genes in human genome are spliced in alternative ways to generate more than one protein per gene. Some examples of alternatively spliced pre-mRNA are: troponin, tropomyosin, myosin, actin, fibronectin, fibrinogen, nerve growth factor, aldolase, alcohol dehydrogenase, calcitonin, SV40 T-antigen, Drosophila sxl, tra and dsx pre-mRNA for sex determination etc.
factors are responsible for activating or repressing the use of processing sites near to where they bind. Thus, the application of SR proteins (serine-arginine rich) and hnRNPs to guide alternative splicing mechanism has been suggested.
(
transcript is altered is called RNA editing. RNA editing, like RNA splicing, is a process in which sequence of RNA changes after or during its transcription i.e. at the level of mRNA. In this form of RNA processing, the nucleotide sequence of the primary transcript is altered by changing / inserting / deleting residues at specific points along the molecule. Thus, the protein produced upon translation is different from that predicted from the gene sequence i.e. coding sequence in RNA differs from the sequence of DNA from which it was transcribed. This is thus a method for increasing protein diversity, similar to alternative splicing. RNA editing occurs in two different situations, with different causes.
$ Site-specific deamination: In mammalian cells, there are cases in wh
occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded. For eg., apolipoprotein B gene and mRNA in mammalian intestine and liver, glutamate receptors in rat brain etc.