Using DNA Subway in the Classroom Red Line - Splicing

DNA Subway The exons and introns of a particular gene get shuffled to create multiple isoforms of a particular protein First demonstrated in the late 1970’s in adenovirus Fairly well characterized in animals (at least somewhat better than in plants) Contributes to protein diversity Affects mRNA stability Alternative splicing review

DNA Subway Alternative splicing in metazoans Human splicing statistics The Alternative Splicing Gallery (ASG): bridging the gap between genome and transcriptome Nucleic Acids Research, 2004, Vol. 32, No. 13 Estimating rates of alternative splicing in mammals and invertebrates. NATURE GENETICS VOLUME 36 | NUMBER 9 | SEPTEMBER 2004 Alternative splicing is well characterized in animals In humans, the vast majority of genes have multiple spliceforms Estimates of up to 80% of human genes are alternatively spliced

DNA Subway Alternative splicing in disease By virtue of its widespread involvement in most of the genomic landscape, AS is important in almost all gene families AS (or mis-splicing) is a very important component of genetic diseases

Mechanisms of splicing

Genome Pre-mRNA E1 E2 I1 E3 I2 E4 Spliced mRNA E1 E2 E3 E4

The majority of AS events have not been functionally characterized DNA Subway Alternative splicing discovery in plants Alternative splicing of RuBisCo was one of the first examples of AS in plants “The data presented here demonstrate the existence of alternative splicing in plant systems, but the physiological significance of synthesizing two forms of rubisco activase remains unclear. However, this process may have important implications in photosynthesis. if these polypeptides were functionally equivalent enzymes in the chloroplast, there would be no need for the production of both polypeptides, and alternative splicing of the rubisco activase mRNA would likely become a dispensable process.” The majority of AS events have not been functionally characterized

5’ Splice Site Pre-mRNA E1 I1 E2 3’ Splice Site Reddy, S.N. Annu. Rev. Plant Biol. 2007 58:267-94 In Arabidopsis out 1470 of 1588 predicted splice sites follow the canonical (GT…AG , CG…AG, AT…AC )consensus sites. (The Plant Journal (2004) 39, 877–885 Intron retention is a major phenomenon in alternative splicing in Arabidopsis)

5’ Splice Site S S Pre-mRNA UTR E1 I1 E2 UTR 3’ Splice Site ATG ATG S S Pre-mRNA UTR E1 I1 E2 UTR 3’ Splice Site - Alternative splicing can effect the entire pre-mRNA transcript (UTRs included) - Alternative splicing can also alter start codons or lead to premature termination codons Mature mRNA m7G UTR E1 E2 UTR AAA...AA

There are 5 main types of splicing Constitutive (familiar/ “normal”) Alternative Donor site Alternative Acceptor site Alternative position Exon Skipping Intron retention m7G UTR E1 E2 UTR AAA...AA

Genome Pre-mRNA E1 E2 I1 E3 I2 E4 Spliced mRNA E1 E2 E3 E4 Constitutive splicing

Pre-mRNA E1 I1 E2 Pre-mRNA E1 E1 I1 E2 Spliced mRNA E1 E2 Alternative donor site (AltD)

Pre-mRNA E1 I1 E2 Pre-mRNA E1 I1 E2 E2 Spliced mRNA E1 E2 Alternative acceptor site (AltA)

Pre-mRNA E1 I1 E2 Pre-mRNA E1 I1 I1 E2 Spliced mRNA E1 E2 Alternative Position (AltP)

Pre-mRNA E1 I1 E2 I2 E3 Pre-mRNA E1 I1 E2 I2 E3 Spliced mRNA E1 E3 Exon skipping (ExonS)

Pre-mRNA E1 I1 E2 Pre-mRNA E1 I1 E2 Spliced mRNA E1 Intron retention (IntronR)

How prevalent are these alternative spliceforms? AS type Events (%) Genes (%) AltD 845 (10.2) 724 (3.3) 1,642 (11.3) 990 (3.2) AltA 1,810 (21.9) 1,452 (6.7) 2,201 (15.1) 1,698 (5.5) AltP 308 (3.7) 200 (0.9) 921 (6.3) 562 (1.8) ExonS 666 (8.1) 379 (1.8) 2,004 (13.8) 999 (3.2) IntronR 4,635 (56.1) 3,094 (14.3) 7,774 (53.5) 4,513 (14.6) Total 8,264 4,707 (21.8) 14,542 6,568 (21.2) Genomewide comparative analysis of alternative splicing in plants PNAS May 2, 2006 vol. 103 no. 18 7175-7180 21,641 genes and Arabidopsis and 30,917 genes in rice were interrogated for Alternative splicing events. An estimated 1/5th of plant genes undergo alternative splicing

Alternative splicing is far less common in plants AS type Arabidopsis Rice Maize Human AltD 3% 11% 5% 42% AltA 18% 22% 24% ExonS 38% 34% 25% IntronR 41% 33% 35% 9% Genome-wide analyses of alternative splicing in plants: Opportunities and challenges Genome Res. 2008. 18:1381-1392 In humans up to 80% of genes undergo AS (compared to ~20% in plants) The types of AS varies across species Intron retention is the most common type of AS in plants

The plant spliceosome is less well characterized than metazoan mechanisms. Plants share similar splice site configurations with animals, but there are significant differences in intron size and composition Reddy, S.N. Annu. Rev. Plant Biol. 2007 58:267-94

How are AS events detected? Splicing in disease: disruption of the splicing code and the decoding machinery. doi:10.1038/nrg2164 High-througput detection is largely based on microarray data provided by cDNA and EST data; now also RNA-Seq PCR based assays

Biological importance of AS So far, AS has been implicated in a number of biologically important roles including: Splicing Transcriptions Flowering regulation Disease resistance Enzymatic activity A database of AS genes is available at plantgdb.org/ASIP/

Some examples: Disease resistance in tobacco In tobacco, the N gene confers resistance to Tobacco Mosaic Virus (TMV) There are two alternative transcripts Ns and NL (short and long) NL lacks 13 of the 14 LRRs that make are a part of the Ns protein Infection with TMV causes NL to become more abundant after infection Expression of Ns in transgenic plants does not confer TMV resistance

Some examples: Jasmonate signaling in Arabidopsis Jasmonate (plant hormone) is involved in cell division and growth, reproduction as well as defense against insects, pathogens, and abiotic stress. AS isoforms (10.4 and 10.3) result in various phenotypic effects (e.g. male sterility, insensitivity to jasmonate inhibition of root growth, etc.)

Some examples: Jasmonate signaling in Arabidopsis The JAZ10.3 isoform results in a premature stop codon in the D exon. The JAZ10.4 (AltD) isoform results in a truncation of the D exon, which leads to the elimination of an important domain (Jas).