Ejercicios de relajación muscular.

3.2.1 Alternative Splicing of pdr-1

Alternative splicing has an important role in expanding protein diversity. Differential splicing of the parkin gene has been observed not only in different organisms, but

also in different human tissues or cell types, and is suggested to be physiologically relevant (Dagata and Cavallaro, 2004; Kitada et al., 2000; Sunada et al., 1998).

Figure 14. pdr-1 Splicing Variants and Protein Isoforms.

Top: C. elegans splice variants. I: full-length pdr-1 (1158 bp); II: in-frame (∆ bp 321-784); III: out-of-

frame (∆ bp 649-740); IV: out-of-frame (45 bp insertion after position 69, 4 bp insertion after position 215, and ∆ bp 649-740); V: out-of-frame (4 bp insertion after position 215). Bottom: C. briggsae splice

variants. I: full-length pdr-1 (1155 bp); II: in-frame (∆ bp 301-645); III: in-frame (∆ bp 70-645); IV: out-

of-frame (∆ bp 454-645, insertion of 8 bp after position 736). Asterisks indicate premature stops, followed by un-translated regions (hatch boxes). Black shading depicts newly spliced coding regions, grey shading shift into another reading frame. Scale bar 0.1kb.

By RT-PCR on total RNA, five different pdr-1 splice variants were identified

from C. elegans, and another four of the C. briggsae homolog (Figure 14). The

cid compositions, and most important, different molecular architectures. Some of the nematode pdr-1 splice

already identified parkin transcripts, others

only present a subset and the total number might be far greater, but one cannot exclude different evolutionary conservation for some parkin splice variants, as well.

Alternative splicing of parkin could potentially generate a large number of

protein isoforms that might impart different properties on the cells displaying them. Furthermore, the expression of the different isoforms could be differentially affected by mutations of the parkin gene. This might provide an explanation for the broad

spectrum of phenotypic abnormalities observed in AR-JP patients.

3.2.2 pdr-1 Transcription is Developmentally Regulated

To analyze the temporal expression pattern of pdr-1, Northern blot analyses were

performed using total RNA from each developmental stage of C. elegans wild type

animals. pdr-1 transcription becomes active in embryogenesis and is maintained

throughout all developmental stages until adulthood (Figure 15).

Figure 15. pdr-1 is Developmentally Regulated.

Northern blot analyses show co-transcriptional regulation of pdr-1 and K08E3.8 during all developmental stages, from embryogenesis (eggs) throughout larval stages (L1-L4) until adulthood (adult). pdr-1 and K08E3.8 transcript levels are specifically up-regulated beginning in L2 and strongly

L3. All transcript levels indicated are ung adult levels and were adjusted for encoded PDR-1 isoforms have different amino a

variants do not perfectly resemble the

however are well conserved, even in humans. Minor variations between nematode and mammalian parkin splice variants, certainly arise from different gene structures

and splice sites, which are highly conserved among rat, mouse and human, but distinct in nematodes. However, alternative splice variants detected so far, might re

increasing in relative to yo

Notably, pdr-1 as well as K08E3.8 transcript levels are specifically up-

regulated beginning at the larval L2 stage and strongly increasing at the L3 stage, reachi

ants of a

o ensure proper expression of the reporter gene, this constructs retains the complete genomic context

re 16).

shown. Lines represent genomic sequences co gfp fusions (Ppdr-1::gfp) contain either ~4.0 kb

respectively). In Ppdr-1::gfp::pdr-1 the gfp coding

start to yield a N-terminal tagged GFP::PDR-1 pr

ng a maximum in the adult. These data corroborate the proposed transcriptional co-regulation of PDR-1 and K08E3.8, and additionally suggest development-specific function(s).

3.2.3 pdr-1 in vivo Expression Pattern

The parkin gene has been shown to display a widespread expression, not only in

humans, but also in a variety of other vertebrates and invertebrates (Horowitz et al., 2001; Huynh et al., 2001; Solano et al., 2000; Stichel et al., 2000).

To determine the expression pattern of C. eleganspdr-1 in vivo different green

fluorescent protein (gfp) reporter constructs were generated. Two vari

promoter gfp construct (Ppdr-1::gfp long and short) were generated, containing either

4.0 kb or at least 650 bp of upstream sequence, fusing the pdr-1 start codon to the gfp coding region (plasmids pBY1013 and pBY1909, respectively) (Figure 16). In

addition, to identify the subcellular localization of PDR-1, a translational fusion construct Ppdr-1::gfp::pdr-1 (plasmid pBY1794) was generated. T

of the operon, including both genes of the transcriptional unit (Figu

Figure 16. pdr-1 Reporter Constructs.

Position and extent of the gfp reporter constructs (Ppdr-1::gfp and Ppdr-1::gfp::pdr-1) relative to pdr-1 are

ntained in the different reporter constructs. Promoter or 650 bp upstream sequence (long and short, sequence is fused in-frame to the pdr-1 translational otein.

The engineered pdr-1 reporter constructs were microinjected into N2 wild type worms

obtain stable lines of transgenic animals, expressing the gfp fusions from

to

extrachromosomal arrays (Figure 17).

Figure 17. C. elegans pdr-1 is Ubiquitously Expressed.

ransgenic expression of different pdr-1::gfp reporter constructs in N2 wild type animals. (A) Embryo xpressing gfp in almost all cells. (B) L2 larval gfp expression in pharyngeal and anal muscles (closed as rve cord (open arrows). (C) L3 larval gfp expression in almost all tissues of adult worms. (D) Cell bodies (open

T e

arrows) as well in neurons of the ventral ne hypodermal cells. (D-J) gfp expression in

arrows) and processes of head neurons. (E) Cytoplasmatic localization of GFP::PDR-1 in a neuron. (F) Pharyngeal muscles (closed arrow) and neurons of the head (open arrows). (G) Body-wall

muscles. (H) Vulval muscles (ventral view). (I) Vulval muscles (lateral view). The vulva opening is

marked by an asterisk.(J) Gonadal gfp expression.

Although mosaic in individual worms, gfp expression patterns were almost identical in

all 12 independent transgenic strains examined In total, 12 independent transgenic lines were analyzed, six for the promoter constructs pBY1013 and pBY1909 (strains BR1948 and BR3187-91, respectively), as well as another six for the translational fusion construct pBY1794 (strains BR3045-50).

In vivo analysis of the gfp reporter constructs confirmed the temporal

expression pattern of pdr-1 observed in transcriptional analyses. GFP signals were

detected from embryogenesis (Figure 17A) throughout all developmental stages (Figure 17B and C) until adulthood (Figure 17D-J). GFP::PDR-1 is highly expressed in most neurons of the head, the tail and the nerve cords, localizing to cell bodies as well as to processes (Figure 17B, 17E-F). GFP staining is mostly cytoplasmic and mainly excluded from the nucleus (Figure 17E). Furthermore, GFP signal was observed in all muscle cells (Figure 17B and 17F-I), as well as in a variety of other tissues, like hypodermal cells (Figure 17C) and gonads (Figure 17J), as well as spermatheca and intestine.

Noteworthy, it appeared that both promoter constructs, although injected at the same concentration as the translational fusion construct, showed slightly stronger GFP signal, suggesting a regulation of PDR-1 at the protein level, most likely by degradation. However, PDR-1 is enriched in neurons and muscles, but present in almost all tissues of the animal, and so conceivably plays an important role in all cells.