Splicing RNA: Mechanisms

Splicing of Group I and II introns Introns in fungal mitochondria, plastids, Tetrahymena pre-rRNA Group I Self-splicing Initiate splicing with a G nucleotide Uses a phosphoester transfer mechanism Does not require ATP hydrolysis. Group II self-splicing Initiate splicing with an internal A Does not require ATP hydrolysis

Self-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981 Exon 1 Exon 2 Intron 1 + pre-rRNA Spliced exon Intron circle Intron linear Nuclear extract GTP + - Products of splicing were resolved by gel electrophoresis: Additional proteins are NOT needed for splicing of this pre-rRNA! Do need a G nucleotide (GMP, GDP, GTP or Guanosine).

Self-splicing by a phosphoester transfer mechanism OH Intron 1 U U P P U A Exon 1 Exon 2 P P G U Exon 1 P P + Intron 1 Exon 2 N15 N16 G A P P G P OH N15 G A OH P + Circular intron

A catalytic activity in Group I intron Self-splicing uses the intron in a stoichiometric fashion. But the excised intron can catalyze cleavage and addition of C’s to CCCCC

Group I intron catalyzes cleavage and nucleotide addition

The intron folds into a particular 3-D structure Has active site for phosphoester transfer Has G-nucleotide binding site

Active sites in Group I intron self-splicing

Domains of the Group I intron ribozyme

Exon exchange via trans-splicing Although splicing is usually in cis, splicing can occur in trans (between 2 different RNAs). The internal guide sequence is only needed for specificity, NOT for catalysis Thus any RNA can serve as an IGS Can engineer RNAs with an IGS complementary to the region 5’ to a mutation for exon exchange. This a potential approach to therapy for some genetic diseases.

RNAs that function as enzymes RNase P Group I introns Group II introns rRNA: peptide bond formation Hammerhead ribozymes: cleavage snRNAs involved in splicing

Hammerhead ribozymes A 58 nt structure is used in self-cleavage The sequence CUGA adjacent to stem-loops is sufficient for cleavage

Design hammerhead ribozymes to cleave target RNAs Potential therapy for genetic disease.

Mechanism of hammerhead ribozyme The folded RNA forms an active site for binding a metal hydroxide Abstracts a proton from the 2’ OH of the nucleotide at the cleavage site. This is now a nucleophile for attack on the 3’ phosphate and cleavage of the phosphodiester bond.

Phosphotransfers for Group I vs. Group II & pre-mRNA 2’ G HO 3’ Exon 1 Exon 2 Exon 1 Exon 2 A 2’ G OH OH 2’ A Exon 1+2 Exon 1+2 + G + OH Group II and pre-mRNA Group I

Splicing of pre-mRNA The introns begin and end with almost invariant sequences: 5’ GU…AG 3’ Use ATP to assemble a large spliceosome Mechanism is similar to that of the Group II fungal introns: Initiate splicing with an internal A Uses a phosphoester transfer mechanism for splicing

Initiation of phosphoester transfers in pre-mRNA Uses 2’ OH of an A internal to the intron Forms a branch point by attacking the 5’ phosphate on the first nucleotide of the intron Forms a lariat structure in the intron Exons are joined and intron is excised as a lariat A debranching enzyme cleaves the lariat at the branch to generate a linear intron Linear intron is degraded

Splicing of pre-mRNA, step 1

Splicing of pre-mRNA, step 2

Investigation of splicing intermediates In vitro splicing reaction: nuclear extracts + ATP+ labeled pre-mRNA Resolve reaction intermediates and products on gels. Some intermediates move slower than pre-mRNA. Suggest they are not linear. Use RNase H to investigate structure of intermediate. RNase H cuts RNA in duplex with RNA or DNA.

RNase H + oligonucleotides complementary to different regions give very different products

Analysis reveals a lariate structure in inter-mediate

Involvement of snRNAs and snRNPs snRNAs = small nuclear RNAs snRNPs = small nuclear ribonucleoprotein particles Antibodies from patients with the autoimmune disease systemic lupus erythematosus (SLE) can react with proteins in snRNPs Sm proteins Addition of these antibodies to an in vitro pre-mRNA splicing reaction blocked splicing. Thus the snRNPs were implicated in splicing

snRNPs U1, U2, U4/U6, and U5 snRNPs Have snRNA in each: U1, U2, U4/U6, U5 Conserved from yeast to human Assemble into spliceosome Catalyze splicing Sm proteins bind “Sm RNA motif” in snRNAs 7 Sm proteins: B/B’, D1, D2, D3, E, F, G Each has similar 3-D structure: alpha helix followed by 5 beta strands Sm proteins interact via beta strands, may form circle around RNA

Sm proteins may form ring around snRNAs ANGUS I. LAMOND Nature 397, 655 - 656 (1999) RNA splicing: Running rings around RNA

Predicted structure of assembled Sm proteins 4th beta strand of one Sm protein interacts with 5th beta strand of next. Channel for single strand of RNA ANGUS I. LAMOND Nature 397, 655 - 656 (1999) RNA splicing: Running rings around RNA

Assembly of spliceosome The spliceosome is a large protein-RNA complex in which splicing of pre-mRNAs occurs. snRNPs are assembled progressively into the spliceosome. U1 snRNP binds (and base pairs) to the 5’ splice site U2 snRNP binds (and base pairs) to the branch point U4-U6 snRNP binds, and U4 snRNP dissociates U5 snRNP binds Assembly requires ATP hydrolysis Assembly is aided by various auxiliary factors and splicing factors.

Spliceosome assembly and catalysis HO 2’ U2 snRNP A HO 2’ U1 snRNP Exon 1 A HO 2’ Exon 2 U5 snRNP U4/U6 U6 U4 Sm proteins snRNAs Other proteins A G U O H U4? A O 2’ G U U6 U2 H U1 U5 Spliceosome Exons 1+2 2’ A

Catalysis by U6/U2 on branch oligonucleotide in vitro Figure 1 Base-pairing interactions in the in vitro-assembled complex of U2–U6 and the branch oligonucleotide (Br). Shaded boxes mark the invariant regions in U6 and previously established base-paired regions are indicated. Dashed lines connect psoralen-crosslinkable nucleotides (S.V. and J.L.M., unpublished data). The circled residues connected by a zigzag can be crosslinked by ultraviolet light. The underlined residues in Br constitute the yeast branch consensus sequence. Asterisks denote the residues involved in the covalent link between Br and U6 in RNA X (see text). Arrowheads point to residues involved in a genetically proven interaction in yeast22. Numbers indicate nucleotide positions from the 5' ends of full-length human U2 and U6. Nature 413, 701 - 707 (2001) Splicing-related catalysis by protein-free snRNAs SABA VALADKHAN & JAMES L. MANLEY

Biochemical results for U6/U2 reaction on Branch oligonucleotide Nature 413, 701 - 707 (2001) Splicing-related catalysis by protein-free snRNAs SABA VALADKHAN & JAMES L. MANLEY

RNA editing RNA editing is the process of changing the sequence of RNA after transcription. In some RNAs, as much as 55% of the nucleotide sequence is not encoded in the (primary) gene, but is added after transcription. Examples: mitochondrial genes in trypanosomes and Leishmania. Can add, delete or change nucleotides by editing

Addition of nucleotides by editing Uses a guide RNA that is encoded elsewhere in the genome Part of the guide RNA is complementary to the mRNA in vicinity of editing U nt at the the 3’ end of the guide RNA initiates a series of phosphoester transfers that result in insertion of that U at the correct place. More U’s are added sequentially at positions directed by the guide RNA Similar mechanism to that used in splicing

What is a gene? Making a correctly edited mRNA requires one segment of DNA to encode the initial transcript and a different segment of DNA to encode each guide RNA. Thus making one mRNA that uses 2 guide RNAs requires 3 segments of DNA - is this 3 genes or 1 gene? Loss-of-function mutations in any of those 3 DNA segments result in an nonfunctional product (enzyme), but they will complement in trans in a diploid analysis! This is an exception to the powerful cis-trans complementation analysis to define genes.

Mammalian example of editing Apolipoprotein B in the intestine is much shorter than apolipoprotein B in the liver. They are encoded by the same gene. The difference results from a single nt change in codon 2153: CAA for Gln in liver, but UAA for termination of translation in intestine The C is converted to U in intestine by a specific deaminating enzyme, not by a guide RNA.