A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing

TS. Kroeger, KP. Watkins, G. Friso, KJ van Wijk, A. Barkan. 2009.  A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing.  Proceedings of the National Academy of Sciences of the United States of America 106: 4537-4532.

Domain of unknown function 860, DUF860, is a protein domain found only in land plants.  One of its proteins WTF1 (“what’s that factor?”), was thought to be required for and promoted the splicing of the introns with which it associates.  To determine which introns WTF1 associates with, the scientists used a genome-wide RNA coimmunoprecipitation assay (RIP-chip).  WTF1 was immunoprecipiated from stroma and combined with fluorescently labeled RNA and  then hybridized to the maize chloroplast genome.  These results were compared to those of the control experiment that used the OE16 antibody which does not bind to RNA.  The array elements that showed association with WTF1 were all from loci containing group II introns, including petB.  To furthur verify these findings, the RNA from the RNA-chip experiment was analyzed with slot-blot hybridization using WTF and OE16.  All of the introns that showed association in the RNA-chip assay, also positively hybridized with WTF1 in the slot-blot.  These experiements showed that the group II introns associated with WTF1 in chloroplasts. Then to determine whether WTF1 promotes splicing in vivo, the splicing of all group II introns was assayed in WTF1 mutants.  They analyzed splicing in WTF1 mutants with moderate ribosome loss, not expected to do much damage as well as with WTF1 mutants with severe ribosome loss.  These results wer compared with the control group and poisened-primer extension assays showed a reduced ratio of spliced to unspliced RNA in the group II introns.  This showed that WTF promotes splicing in the introns with which it associates.

PCR petB

Rico Jordan and I, Earica Johnson, set up 9 PCR reactions mixtures on 9/30/2011.  One mixture was the negative control and the other eight mixtures were pairs of the 4 orchid species used for DNA extraction.  Our task was to amplify the petB region of the chloroplast genome in these 4 orchid species.  On 10/6/2011, I prepared our extracted DNAs for gel electrophoresis by mixing 5ul of the DNAs with 1ul of the loading dye EZ Vision One.  The samples were electrophoresed for approximately 20 minutes, then the gel was removed from the chamber and the image below was obtained.

As demonstrated in the image below, lanes 3-8 yielded highly successful results as evidenced by the thick, single bands.  Even though there are obvious bands in lanes 9 and 10, they are not comparable in size to those in lanes 3-8 and are therefore considered weakly successful.  This result could be due to not obtaining enough DNA during extraction as became apparent during the subsequent purification of this DNA in which only a 30.1ng/uL concentration was obtained by NanoDrop Spectrophotometry.

1% agarose gel with DNAs 3-4, 3-7, 382, 384

Lane	DNA Code	Species or Marker	Success?
1	–	Molecular mass ruler	–
2	–	Negative control	–
3	3-7	Holcoglossum wangii	++
4	3-7	Holcoglossum wangii	++
5	3-4	Plectrelminthus caudutus	++
6	3-4	Plectrelminthus caudutus	++
7	382	Stereochilus hirtus	++
8	382	Stereochilus hirtus	++
9	384	Luisa filiformis	+
10	384	Luisia filiformis	+

Holcoglossum wangii

Kingdom: Plantae  Division: Magnoliophyta  Class: Liliopsida  Order: Asparagales  Family: Orchidaceae  Subfamily: Epidendroideae  Tribe: Vandeae  Subtribe: Aeridinae

Holcoglossum wangii Christenson 1998 is an epiphytic plant, one which grows on another plant or object, found in the coniferous forests of southern China and northern Vietnam.  These plants are found at elevations around 250 to 1200m.  H.wangii grows as an inflorescence, a group of flowers on a stem.  The flowers are small, usually 2in or less, and are white with yellow-lobes and pinkish/purple specks.  The leaves are fleshy and terete (cylindrical and tapering) and comes in two forms: short-leaf (6in) and long-leaf (12-18in).  Mature plants typically reach 10-12in but can flower as small as 4in.  Seedlings are 4-6in.

H.wangii is best grown when mounted on a stick or in a basket.  They require cool to intermediate temperatures and moderate sunlight.  They should be grown in a moist and well drained environment but if hung, the roots should be watered frequently.  They bloom in the fall and winter.

Inflorescence of flowers

Flower of H.wangii

Specimen used for DNA extraction

References:

http://orchids.wikia.com/wiki/Holcoglossum_wangii

http://www.orchidspecies.com/holcowangii.htm

http://www.jlorchids.com/catalog/all_others3.html