On the functional significance of alternative splicing

Alternative splicing – the choice of different splice sites and/or exons in a primary transcript that possesses numerous exons and introns – is a widespread phenomenon.  With the advent of very sensitive as well as high-throughput techniques, it has proven possible to identify alternatively-spliced transcripts for many, perhaps a majority, perhaps all genes.  However, the very sensitivity of the techniques raise the interesting and important question of the functional significance of what is observed.  Thus, it is possible that much (most, all?) of the alternatively-spliced mRNA isoforms are the results of splicing errors.  (Some in the blogosphere are of the opinion that alternative splicing is mostly artifact.)  Accordingly, studies that speak to the functions of the products of alternative splicing are always of interest.

A recent study from Stephen Mount’s lab illustrates an excellent approach to this problem.  In this study, two different isoforms of a so-called SR protein (the Arabidopsis SR45 splicing factor) were studied.  These isoforms are encoded by different alternatively-spliced mRNAs, and differ by eight amino acids that correspond to one of two 3′ splice sites that are chosen in the course of pre-mRNA processing.  Loss-of-function mutant plants that do not make SR45 show a range of developmental phenotypes that affect flowers and roots.  Interestingly, when one isoform is expressed* in a loss-of-function mutant background, the flower phenotype is reversed but not the root phenotype.  Conversely, expression of the other isoform restores normal root growth but not flower morphology.  The bottom line is that the two SR45 isoforms have distinct functions.  Thus, at least in this case, alternative splicing has important roles in growth and development.

The abstract:

The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis thaliana, they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3′ splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific GFP fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both Serine 219 and the adjacent Threonine 218 (SR45.1-T218A+S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions.

The citation:

Zhang, X.-N. and Mount, S. M. 2009.  Two alternatively spliced isoforms of the Arabidopsis thaliana SR45 protein have distinct roles during normal plant development.  Plant Physiology Preview. Published on April 29, 2009; 10.1104/pp.109.138180

Don’t forget to visit Stephen Mount’s web site or his blog.

* – different isoforms can be expressed in the loss-of-function mutant by introducing into this mutant transgenes that express either cDNA; when this is done, the intron, and thus alternative splicing, is removed from the equation.

Finally, for the reader’s information, a random laundry list of similar studies that I posted on Larry Moran’s blog some time ago.  I haven’t included links to the papers, but interested readers should have no problems tracking them down.  The list isn’t exhaustive, but it is enough to drive home the message that alternative splicing does have functional significance.  Enjoy.

Schöning JC, Streitner C, Meyer IM, Gao Y, Staiger D.
Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis.
Nucleic Acids Res. 2008 Dec; 36(22):6977-87

Dinkins RD, Majee SM, Nayak NR, Martin D, Xu Q, Belcastro MP, Houtz RL, Beach CM, Downie AB.
Changing transcriptional initiation sites and alternative 5′- and 3′-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments.
Plant J. 2008 Jul;55(1):1-13.

Puyaubert J, Denis L, Alban C.
Dual targeting of Arabidopsis holocarboxylase synthetase1: a small upstream open reading frame regulates translation initiation and protein targeting.
Plant Physiol. 2008 Feb;146(2):478-91.

Bove J, Kim CY, Gibson CA, Assmann SM.
Characterization of wound-responsive RNA-binding proteins and their splice variants in Arabidopsis.
Plant Mol Biol. 2008 May;67(1-2):71-88.

Bocobza S, Adato A, Mandel T, Shapira M, Nudler E, Aharoni A.
Riboswitch-dependent gene regulation and its evolution in the plant kingdom.
Genes Dev. 2007 Nov 15;21(22):2874-9.

Muralla R, Chen E, Sweeney C, Gray JA, Dickerman A, Nikolau BJ, Meinke D.
A bifunctional locus (BIO3-BIO1) required for biotin biosynthesis in Arabidopsis.
Plant Physiol. 2008 Jan;146(1):60-73.

Zhang XC, Gassmann W.
Alternative splicing and mRNA levels of the disease resistance gene RPS4 are induced during defense responses.
Plant Physiol. 2007 Dec;145(4):1577-87.

Rossignol P, Collier S, Bush M, Shaw P, Doonan JH.
Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing variant of telomerase.
J Cell Sci. 2007 Oct 15;120(Pt 20):3678-87.

Castells E, Puigdomènech P, Casacuberta JM.
Regulation of the kinase activity of the MIK GCK-like MAP4K by alternative splicing.
Plant Mol Biol. 2006 Jul;61(4-5):747-56.

Lee JR, Jang HH, Park JH, Jung JH, Lee SS, Park SK, Chi YH, Moon JC, Lee YM, Kim SY, Kim JY, Yun DJ, Cho MJ, Lee KO, Lee SY.
Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis.
Plant J. 2006 Aug;47(3):457-66.

de la Fuente van Bentem S, Vossen JH, Vermeer JE, de Vroomen MJ, Gadella TW Jr, Haring MA, Cornelissen BJ.
The subcellular localization of plant protein phosphatase 5 isoforms is determined by alternative splicing.
Plant Physiol. 2003 Oct;133(2):702-12.

Savaldi-Goldstein S, Aviv D, Davydov O, Fluhr R.
Alternative splicing modulation by a LAMMER kinase impinges on developmental and transcriptome expression.
Plant Cell. 2003 Apr;15(4):926-38.

Jasinski S, Perennes C, Bergounioux C, Glab N.
Comparative molecular and functional analyses of the tobacco cyclin-dependent kinase inhibitor NtKIS1a and its spliced variant NtKIS1b.
Plant Physiol. 2002 Dec;130(4):1871-82.

Macknight R, Duroux M, Laurie R, Dijkwel P, Simpson G, Dean C.
Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA.
Plant Cell. 2002 Apr;14(4):877-88.

Dinesh-Kumar SP, Baker BJ.
Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance.
Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1908-13.

Zhou DX, Kim YJ, Li YF, Carol P, Mache R.
COP1b, an isoform of COP1 generated by alternative splicing, has a negative effect on COP1 function in regulating light-dependent seedling development in Arabidopsis.
Mol Gen Genet. 1998 Feb;257(4):387-91.