RNA splicing By xiao yi 生物学基地班 200431060010

In most cases of eukaryotic gene, the coding sequences is interrupted by noncoding sequences The coding sequences are called exons The noncoding sequences are called introns

Before translation, the introns of pre-RNA must be removed, and this process is called RNA splicing

RNA splicing: the process by which introns are removed from the pre-mRNA. Alternative splicing : some pre-mRNAs can be spliced in more than one way , generating alternative mRNAs. 60% of the human genes are spliced in this manner.

The chemistry of RNA splicing Sequences within the RNA Determine Where Splicing Occurs The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs.

the exon-intron boundary at the 5’ end of the intron is called 5’ splicing site

Branch point site an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

The intron is removed in a Form Called a Lariat as the Flanking Exons are joined RNA splicing is achieved by two successive transesterification reactions

Step 1 The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5’ splice site. As a result, the 5’ exon is released and the 5’-end of the intron forms a three-way junction structure.

Step 2 The OH of the 5’ exon attacks the phosphoryl group at the 3’ splice site. As a consequence, the 5’ and 3’ exons are joined and the intron is liberated in the shape of a lariat.

Exons from different RNA molecules can be fused by Trans-splicing The only difference is that the other product---the lariat in the standard reaction---is, in trans splicing, is a Y shaped branch structure instead

THE SPLICESOME MACHINERY RNA splicing is carried out by a large complex called spliceosome The spliceosome comprises about 150 proteins and 5 snRNAs Many functions of the spliceosome are carried out by its RNA components.

The five RNAs U1, U2, U4, U5, and U6 are called small nuclear RNAs (snRNAs) The complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNP)

Three roles of snRNPs in splicing They recognize the 5’ splicing site and the branch site They bring those site together as required They catalyze (or help catalyze) the RNA cleavage and joining reactions

SPLICING PATHWAYS Assembly Rearrangement catalysis

Assembly step 1 1. U1 recognize 5’ splice site. 2. One subunit of U2AF binds to Py tract and the other to the 3’ splice site. The former subunits interacts with BBP and helps it bind to the branch point. 3. Early (E) complex is formed

Assembly step 2 1. U2 binds to the branch site, and then A complex is formed. 2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded. This A residue is available to react with the 5’ splice site.

Assembly step 3 1. U4, U5 and U6 form the tri-snRNP Particle. 2. With the entry of the tri-snRNP, the A complex is converted into the B complex.

Assembly step 4 U1 leaves the complex, and U6 replaces it at the 5’ splice site. U4 is released from the complex, allowing U6 to interact with U2 .This arrangement called the C complex.

U1 leaves the complex, and U6 replaces it at the 5’ splice site. U4 is released from the complex, allowing U6 to interact with U2 .This arrangement called the C complex.

Catalysis Step 1 Formation of the C complex produces the active site, with U2 and U6 RNAs being brought together Formation of the active site juxtaposes the 5’ splice site of the pre-mRNA and the branch site, allowing the branched A residue to attack the 5’ splice site to accomplish the first transesterfication reaction.

Catalysis Step 2: U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3’-OH of the 5’ exon attacks the 3’ splice site.

Self-splicing introns reveal that RNA can catalyze RNA splicing TABLE 13-1 Three class of RNA Splicing Class Abundance Mechanism Catalytic Machinery Nuclear pre-mRNA Very common; used for most eukaryotic genes Two transesterification reactions; branch site A Major spliceosome Group II introns Rare; some eu-Karyotic genes from organelles and prokaryotes Same as pre-mRNA RNA enzyme encoded by intron (ribozyme) Group I introns Rare; nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genes Two transesterific-ation reactions; exogenous G Same as group II introns

When we examine the group 1 and group 2 self-splicing, we find the intron itself folds into a specific conformation within the precursor RNA and catalyze the chemistry of its own release

The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.

Group I introns release a linear intron rather than a lariat Instead of a branch point A residue, they use a free G nucleotide or nucleoside. This G species is bound by the RNA and its 3’ OH group is presented to the 5’ splicing site. Here fuses the G to the 5’ end of the intron. The freed 3’ end attacks the 3’ splicing site. In this case the intron is linear rather than a lariat structure

Two kinds of splice-site recognition errors Splice sites can be skipped. “Pseudo” splice sites could be mistakenly recognized, particularly the 3’ splice site.

Two ways to enhance the accuracy RNA polymerase carries with it various proteins with roles in RNA processing SR (serine argenine anthentic) proteins bind to sequences to called exonic splicing enhancers (ESEs) within the exons

SR proteins are essential for splicing They ensure the accuracy and efficiency of constitutive splicing. They also regulate alternative splicing They come in many varieties, some controlled by physical signals, others constitutively active. Some are expressed preferentially in certain cell types and control splicing in cell-type specific patterns.

Alternative splicing RNAs can be spliced in alternative ways to generate different mRNAs and, thus, different protein products

There are five ways to splice a RNA

Alternative splicing can be either constitutive or regulated Constitutive alternative splicing: more than one product is always made from a pre-mRNA Regulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types

Alternative splicing is regulated by activators and repressors Proteins that regulate splicing bins to specific sites called exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS) The former enhance and the latter repress, splicing at nearby splice sites

The SR proteins bind RNA using one domain----RNA-recognition motif (RRM) Each SR protein use RS domain which is rich in arginine and serine to mediate interactions between the SR protein and proteins within the splicing machinery

Heterogeneous nuclear ribonucleoprotein (hnRNP) bind to RNA but lack the RS domain so cannot recruit splicing machinary, instead, they repress the use of those sites

hnRNPI protein repress splicing by two ways: hnRNPI bind to each end of exon, then interact with each other, looping out the exon hnRNPI coat the RNA across the whole exon, making the exon invisible to the splicing machinary

A small group of intron are spliced by minor spliceosome This spliceosome works on a minority of exons, and those have distinct splice-site sequence. The chemical pathway is the same as the major spliceosome.

Exon shuffling Exons are shuffled by recombination to produce gene encoding new proteins

1. Introns early model – introns existed in all organisms but have been lost from bacteria. 2. Intron late model – introns never existed in bacteria but rather arose later in evolution.

Why have the introns been retained in eukaryotes? 1. The need to remove introns, allows for alternative splicing which can generate multiple proteins from a single gene. 2. Having the coding sequence of genes divided into several exons allows new genes to be created by reshuffling exon.

Three observations suggest exon shuffling actually occur 1. The borders between exons and introns within a gene often coincide with the boundaries between domains within the protein encoded by that gene.

2. Many genes, and proteins they encode, have apparently arisen during evolution in part via exon duplication and divergence.

3. Related exons are sometimes found in unrelated genes.

RNA EDITING RNA editing is another way of changing the sequence of an mRNA

1. Site specific deamination : 1. A specifically targeted C residue within mRNA is converted into U by the deaminase. 2. The process occurs only in certain tissues or cell types and in a regulated manner.

3. Adenosine deamination also occurs in cells 3. Adenosine deamination also occurs in cells. The enzyme ADAR (adenosine deaminase acting on RNA) convert A into Inosine. Insone can base-pair with C, and this change can alter the sequence of the protein. 4. An ion channel expressed in mammalian brains is the target of Adenosine deamination.

2 Guide RNA-directed uridine insertion or deletion. 1. This form of RNA editing is found in the mitochondria of trypanosomes. 2. Multiple Us are inserted into specific region of mRNAs after transcription (or US may be deleted).

3. The addition of Us to the message changes codons and reading frames, completely altering the “meaning” of the message. 4. Us are inserted into the message by guide RNAs (gRNAs) .

Guide RNAs gRNAs range from 40 to 80 nucleotides in length and are encoded by genes distinct from those that encode the mRNAs they act on

gRNAs have three regions: 1.Anchor 2.Editing region 3.poly-U stretch

mRNA transport Once processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translation

Movement from the nucleus to the cytoplasm is an active and carefully regulated process. The damaged, misprocessed and liberated introns are retained in the nucleus and degraded.

A typical mature mRNA carries a collection of proteins that identifies it as being ready for transport. Export takes place through the nuclear pore complex. Once in the cytoplasm, some proteins are discarded and are then imported back to the nucleus for another cycle of mRNA transport. Some proteins stay on the mRNA to facilitate translation.

Thank you By xiao yi 200431060010