CHAPTER 13 RNA Splicing Made by Ren Jun ( 任鋆 生物科学 )

RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA.

RNA processing events include : capping of the 5’ end of the RNA; splicing; and polyadenylation of the 3’ end of the RNA.

The coding sequence of a gene is contiguous in the vast majority of cases in bacteria and their phage. However, many eukaryotic genes are mosaics, consisting of blocks of coding sequences (exons) separated from each other by blocks of noncoding sequences (introns).

The primary transcript for a typical eukaryotic gene contains introns as well as exons. Introns must be removed before translation. The process that introns are removed from the pre-mRNA is called RNA splicing, occurring with great precision. Some pre-mRNAs can be spliced in more than one way, generating alternative mRNAs. This is called alternative splicing.

OUTLINE The Chemistry of RNA Splicing The Spliceosome Machinery Splicing Pathways Alternative Splicing Exon Shuffling RNA Editing mRNA Transport

TOPIC 1 The Chemistry of RNA Splicing

Sequences within the RNA Determine Where Splicing Occurs 5’ splice site 3’ splice site branch point site Splice sites are the sequences immediately surrounding the exon-intron boundaries.

GT-AG rule describes the presence of these constant dinucleotides at the first two and last two positions of introns of nuclear genes.

The Intron Is Removed in a Form Called a Lariat as the Flanking Exons Are Joined two successive transesterification reactions The 2’OH of the conserved A at the branch site attack the phosphoryl group of the conserved G in the 5’ splice site three-way junction The newly liberated 3’OH of the 5’ exon attacks the phosphoryl group at the 3’ splice site intron lariat

the first the second three-way junction

Features of pre-mRNA splicing: It takes place in the nucleus, before the mature mRNA can be exported to the cytoplasm. It requires a set of specific sequences. It takes places in a two-step reaction, snRNPs are involved. The final step is methylation on the N6 position of A residues particularly in the sequence 5’-RRACX-3’.

Exons from Different RNA Molecules Can Be Fused by Trans-Splicing In alternative splicing, exons can be skipped Trans-splicing gets two exons carried on different RNA molecules spliced together forming a Y- shaped branch structure (not a lariat!)

TOPIC 2 The Spliceosome Machinery

The transesterification reactions are mediated by the spliceosome comprise about 150 proteins and 5 RNAs The five RNAs U1,U2,U4,U5,U6 small nuclear RNAs (snRNAs) The RNA-protein complexes small nuclear ribonuclear proteins (snRNPs) Many of the functions of the spliceosome are carried out by its RNA components rich in uracil

The snRNPs have three roles in splicing recognize the 5’ splice site and the branch site bring those sites together as required catalyze (or help to catalyze) the RNA cleavage and joining reactions Interactions are important to perform these functions!

TOPIC 3 Splicing Pathways

Assembly, Rearrangement, and Catalysis Within the Spliceosome The 5’ splice site is recognized by the U1 snRNP.

One subunit of U2AF binds to the Py tract and helps BBP (branch-point binding protein) bind to the branch site, and the other to the 3’ splice site.  The Early (E) complex is formed.

U2 snRNP binds to the branch site, aided by U2AF and displacing BBP. A complex

The U4 and U6 snRNPs, along with the U5 snRNP (the tri-snRNP particle), join the A complex and convert it into the B complex.

U6 replaces U1 at the 5’ splice site.  The above steps complete the assembly pathway.

U4 is released from the complex, allowing U6 to interact with U2.  This arrangement forms the C complex and produces the active site. The 5’ splice site and the branch site are juxtaposed. the first transesterification reaction The U5 snRNP helps to bring the two exons together, triggering the second reaction. Finally, the mRNA product and the snRNPs are released.

U6-U4 pairing is incompatible with U6- U2 pairing. When U6 joins the spliceosome it is paired with U4. Release of U4 allows a conformational change in U6; one part of the released sequence forms a hairpin (dark grey), and the other part (black) pairs with U2. Because an adjacent region of U2 is already paired with the branch site, this brings U6 into juxtaposition with the branch.

assembly catalysis E complex A complex B complex C complex

Two points should be noticed: Some of the components of the splicing machinery do not arrive or leave precisely when described above. It is also not possible to be sure of the order of some changes. The tragedy of forming the active sited ensures the correct splicing

Self-splicing Introns Reveal that RNA Can Catalyze RNA Splicing Self-splicing introns are ones that fold into a specific conformation within the precursor RNA and catalyze the chemistry of its own release They can remove themselves from RNAs in the test tube in the absence of any proteins or other RNA molecules

grouped into two classes on the basis of structure and splicing mechanism Strictly speaking, self-splicing introns are not enzymes. (Why?) The mechanism of group Ⅱ introns is the same as nuclear pre-mRNAs. (a highly reactive Adenine within the intron) Table 13-1 gives the comparison of three classes of RNA splicing

Group Ⅰ Introns Release a Linear Intron Rather than a Lariat use a free G nucleotide or nucleoside instead of a branch point A residue This G species is bound by the RNA and its 3’OH group is presented to the 5’ splice site. Two steps of the reaction are similar to that of pre-mRNA. (But the result is a linear intron with the “G” fused to the 5’ end of it.)

Some characteristics of group Ⅰ introns smaller than group Ⅱ introns a conserved secondary structure  a binding pocket that accommodates the G ribonucleotide or ribonucleoside  an “internal guide sequence” that base-pairs with the 5’ splice site sequence Box 13-1 tells us how group Ⅰ introns can be converted into true ribozymes

Group Ⅰ introns have a common secondary structure that is formed by 9 base paired regions. The sequences of regions P4 and P7 are conserved, and identify the individual sequence elements P, Q, R, and S. P1 is created by pairing between the end of the left exon and the IGS of the intron; a region between P7 and P9 pairs with the 3' end of the intron.

Much of the sequence of a self- splicing intron is critical for the splicing reaction It must fold into a precise structure to perform the reaction chemistry In vivo, the intron is complexed with a number of proteins that help stabilize the correct structure—partly by shielding regions of the backbone from each other

Some views related to evolution The similar chemistry seen in self- and spliceosome-mediated splicing is believes to reflect an evolutionary relationship Perhaps ancestral group Ⅱ -like self- splicing introns were the starting point for the evolution of modern pre-mRNA splicing

How Does the Spliceosome Find the Splice Sites Reliably? Although there exists one mechanism that guards against inappropriate splicing, errors may happen.  First, splice sites can be skipped  Second, other sites, close in sequence but not legitimate splice sites, could be mistakenly recognized (“pseudo” splice site)

But we also have two ways in which the accuracy of splice-site selection can be enhanced The co-transcriptional loading process greatly diminishes the likelihood of exon skipping. (While transcribing a gene to produce the RNA, RNA polymerase Ⅱ carries with it various proteins with roles in RNA processing)

A second mechanism ensures that splice sites close to exons are recognized preferentially  Serine Argenine rich (SR) proteins bind to sequences called exonic splicing enhancers (ESEs) within the exons  interact with components of the splicing machinery, recruiting them to the nearby splice sites  specifically recruit the U2AF proteins to the 3’ splice site and U1 snRNP to the 5’ site (These factors demarcate the splice sites for the rest of the machinery to assemble correctly)

ensure the accuracy and efficiency of constitutive splicing regulate alternative splicing come in many varieties SR proteins are essential for splicing

TOPIC 4 Alternative Splicing

Single Genes Can Produce Multiple Products by Alternative Splicing Many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products

Alternative splicing can arise by a number of means

Since we have mechanisms take ensure variations of this sort do not take place, how does alternative splicing occur so often? Some splice sites are used only some of the time, leading to the production of different versions of the RNA from different transcripts of the same gene

Alternative splicing can be either constitutive or regulated Constitutive alternative splicing always makes more than one product from the transcribed gene In the case of regulated splicing, different forms are generated at different times, under different conditions, or in different cell or tissue types

constitutive alternative splicing: T antigen of the monkey virus SV40

Alternative Splicing Is Regulated by Activators and Repressors exonic splicing enhancer (ESE) intronic splicing enhancer (ISE) exonic splicing silencer (ESS) intronic splicing silencer (ISS)

important in directing the splicing machinery to many exons 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

Structure of the SR proteins RNA-recognition motif (RRM) bind RNA RS domain (rich in arginine and serine, found at the C-terminal end) mediate interactions between the SR protein and proteins within the splicing machinery and recruit it to a nearby splice site

Most silencers are recognized by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family lack the RS domains so cannot recruit the splicing machinery block specific splice sites and repress the use of these sites cooperative and competitive binding

inhibition of splicing by hnRNPI bind at each end of the exon and conceal it within a loop coat the entire exon

The meaning of alternative splicing Multiple protein products can be produced from a single gene (isoforms) used simply as a way of switching expression of the gene that encodes only a single functional protein on and off  determine whether or not an exon with the stop codon is included in a given mRNA  regulate the use of an intron related to mRNA transport

A Small Group Of Introns Are Spiced by an Alternative Spliceosome Composed of a Different Set of snRNPs In higher eukaryotes some pre-mRNAs are spliced by a low-abundance form of spliceosome The minor spliceosome recognizes rarely occurring introns having consensus sequences (AU-AC termini) the same chemical pathway

U11 and U12 components have the same roles as U1 and U2 of the major form U4 and U6 share the same names but are distinct The U5 component is identical

TOPIC 5 Exon Shuffling

Two likely explanations for the situation that introns are almost nonexistent in bacteria introns early model: introns existed in all organisms but have been lost from bacteria introns late model: introns never existed in bacteria but rather arose later in evolution

Why have the introns been retained in eukaryotes and in the extensive form in multicellular eukaryotes? The presence of introns, and the need to remove them, allows for alternative splicing Having the coding sequence of genes divided into several exons allows new genes to be created by reshuffling exons

Three observations strongly suggest that the reshuffling process actually occurs 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 duplication and divergence Related exons are sometimes found in otherwise unrelated genes

Exons Are Shuffled by Recombination to Produce Genes Encoding New Proteins The size ratio ensures that recombination is more likely to occur within the introns than within the exons (Thus…) The mechanism of splicing guarantees that almost all recombinant genes will be expressed

TOPIC 6 RNA Editing

The sequence of the primary transcript is altered by either changing, inserting or deleting residues at the specific points along the molecules is called RNA editing.

RNA Editing Is Another Way of Altering the Sequence of an mRNA two mechanisms that mediate editing site-specific deamination guide RNA-directed uridine insertion or deletion

Site-specific deamination A specifically targeted cytosine residue within mRNA is converted into uridine by cytidine deaminase (occur only in certain tissues or cell types and in a regulated manner)

The longer form of apolipoprotein B, found in the liver, is involved in the transport of endogenously synthesized cholesterol and triglycerides. The smaller version, found in the intestines, is involves in the transport of dietary lipids to various tissues.

Adenosine deamination carried out by the enzyme ADAR (adenosine deaminase acting on RNA) produces Inosine that can base- pair with cytosine. This change can readily alter the sequence of the protein encoded by the mRNA

Editing of mRNA occurs when a deaminase acts on an adenine in an imperfectly paired RNA duplex region.

guide RNA-directed uridine insertion or deletion

Three regions of guide RNA (gRNA) At the 5’ end is the “anchor” and directs the gRNA to the region of the mRNA it will edit The middle determines exactly where the Us will be inserted within the edited sequence At the 3’ end is a poly-U stretch

an RNA-RNA duplex with looped out single- stranded regions endonuclease 3’ terminal uridylyl transferase (TUTase) RNA ligase

The biological significance of editing proofreading translation regulation expanded genetic information

TOPIC 7 mRNA Transport

Once Processed, mRNA Is Packaged and Exported from the Nucleus into the Cytoplasm for Translation The movement is not a passive process, and must be carefully regulated A typical mature mRNA carries a collection of proteins that identifies it as being mRNA destined for transport It is the set of proteins, not any individual kind of protein, that marks RNAs for either export or retention in the nucleus

Export takes place through a special structure in the nuclear membrane called the nuclear pore complex mRNAs and their associated proteins require active transport Once in the cytoplasm, the proteins are discarded, and are then recognized for import back into the nucleus Export requires energy supplied by hydrolysis of GTP by a GTPase protein called Ran