Identification and Sequence Homology of the SRP54 gene in Carassius auratus




Patrick T. Dalton

Introduction

Abstract

SRP54 is an important protein subunit of the signal recognition particle (SRP) that helps in the translocation of secretory proteins to the ER membrane. Described below is a probable partial sequence of the SRP54 gene in Carassius auratus (goldfish). This sequence showed a 70% homology to the human sequence reported in GenBank. The isolation of the partial sequence was attained using RNA extraction techniques with RT-PCR and Sanger sequencing. Primers were developed from regions of high homology between various species and a 1370 bp sequence was isolated. The coding region of human SRP54 is 1514 bp therefore the piece that was isolated represents a significant portion of the gene.

Introduction

SRP is an important complex that is associated with protein targeting to the endoplasmic reticulum (ER). It is a cytosolic particle that can transiently bind to an ER signal sequence in a nascent protein, to the large ribosomal subunit, and to the SRP receptor in the ER membrane (1). SRP54 is a 54kD subunit of the SRP. Cross-linking experiments have shown that it can interact directly with the ER signal sequence, as it emerges from the ribosome, in the absence of GTP (2,3,13). SRP54 is an integral component of SRP, which not only interacts with the nascent signal peptide, but also with SRP RNA (4). In human SRP54 there is a region that contains a large number of clustered methionine residues whose hydrophobic side chains are thought to protrude outward and bind to the hydrophobic side chains that form the central core of an ER signal sequence (5).
The way the method works is that when a signal sequence emerges from the ribosome, SRP interacts with it and targets the resulting complex to the ER membrane by binding to the SRP receptor. Specifically, SRP54 binds to the signal sequence when it emerges from the ribosome (6). Following this, SRP releases the signal sequence into the translocation channel (translocon). SRP54 is a GTPase. It has been reported that a ribosomal component promotes GTP-binding to SRP54 (7). GTP-bound SRP54 is required for high affinity interaction between SRP and its receptor in the ER membrane. This interaction induces the release of the signal sequence from SRP, the insertion of the nascent polypeptide chain into the translocon, and GTP hydrolysis. Therefore, upon contact between SRP54 and the SRP receptor, the signal sequence is released, the bound GTP gets hydrolyzed and SRP dissociates from its receptor.
The SRP receptor (SR) is composed of two subunits: a 70kD α-subunit and a 30kD β-subunit. The SRP54 and SRα subunits (also a GTPase) of the SRP and the SR undergo a tightly coupled GTPase cycle that mediates the signal sequence-dependent attachment of ribosomes to the Sec61 complex of the translocon. It is the cooperative binding of GTP to SRP54 and SRα that stabilizes the SRP-SR complex and initiates signal sequence transfer from SRP54 to Sec61α, which is a subunit of the Sec61 complex (8). It has been shown that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocon could only occur when GTP binding to SRα was permitted (2).
The SRP54 gene has been sequenced in the following species (Metazoa): human (9), mouse (10), canine (9, 10), D. Melanogaster (11), C. Elegans (12), and partial sequences are available from rat (10) and zebrafish (GenBank). The coding region of human SRP54 is 1514 bp in length. It has been found to consist of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high amount of methionine residues (M-domain) (13).


Figures


Figure 1-Total RNA product after DNase treatment. 100 bp ladder (Lane 1) and DNase treated RNA extraction product (Lane 2). RNA isolation and DNase treatment was performed according to the Methods and Materials. RNA products were visualized by ethidium bromide staining and run on an agarose gel.


Figure 2-PCR amplification using various primer combinations. PCR amplification was performed as stated in the Methods and Materials. A 1.5 kb faint band was visualized (Lane 1) on the UV bed but cannot be seen in the picture. 100 bp ladder (Lane 9) and actin control (Lane 8). No products appeared in Lanes 2,3,4,5,6 and 7.


Figure 3-Nesting reaction using the 1.5 kb PCR product as a template for various primer combinations. Three bands of predicted size were visualized in this reaction. A 1.4 kb product (Lane 4), a 400 bp product (Lane 6), a 900 bp product (Lane 7). 100 bp ladder (Lane 1). No products appeared in Lanes 2,3, and 5.


Figure 4-Sequence alignment of the derived goldfish SRP54 sequence and the human SRP54 sequence from GenBank. Sequences were aligned using MacVector and the ClustalW Alignment Format.


Results

RNA extraction was performed on the gills of goldfish. The product from the isolation yielded three distinct bands (data not shown). The first band visualized is DNA in the sample. The second band represents 28S RNA and the third band from the top is 18S RNA. Following DNase treatment the first band was eliminated (Fig 1, Lane 2) while the 28S and 18S bands remained. The DNase treated RNA was subjected to RT-PCR. The resulting cDNA was then PCR amplified using various combinations of forward and reverse primers. A faint band, representing the predicted 1.5 kb product, was detected in the reaction using SRP54-1 and SRP54-6 primers (Fig 2, Lane 1). This band does not show up in the picture but was visualized when the gel was placed on a UV bed. An actin positive control was used to verify the template had not degraded and produced a band. The actin control migrated correctly and is approximately 600 bp in length (Fig 2, Lane 8).
The next step taken was to run a nesting reaction, because of the low yield result from the initial PCR amplification. Primers that should sit on the inside of the 1.5 kb PCR product were used in a PCR amplification reaction. The PCR product itself was used as the template in the reaction. Several bands of predicted size were visualized from this PCR amplification (Fig 3). A PCR product approximately 1.4 kb in length was produced using primers SRP54-2 and SRP54-6 (Fig 3, Lane 4). Another PCR product approximately 400 bp in length was produced using primers SRP54-2 and SRP54-4 (Fig 3, Lane 6). Additionally, a third PCR product was produced approximately 900 bp in length, using primers SRP54-2 and SRP54-5 (Fig 3, Lane 7).
A sequencing reaction was then performed as described in the Methods and Materials. The resulting nucleotide sequences were aligned to the human SRP54 sequence from GenBank. The sequences aligned to the human SRP54 sequence at the predicted regions in relation to the primers used. Gaps in the sequence were filled by primer walking, using primers SRP54-7 and SRP54-8. By piecing together the resulting partial sequences, 1370 bps of the derived sequence aligned to the human SRP54 sequence with a 70% homology (Fig 4). The start codon for the human sequence is located at bp 228 while the stop codon is located at bp 1742.

Discussion

There is a relatively high homology between the goldfish SRP54 sequence reported above and the published human sequence. There are still gaps in the beginning and the end of the goldfish sequence that need to be resolved but this should be possible in the future when the zebrafish project has been completed. The partial sequence of the zebrafish that was available for this experiment was only 600 bp and covered the beginning of the coding region. This made it difficult to develop primers that would span the entire coding region. With more time, the unknown bases in the sequencing reaction can be determined. Due to the presence of these unknown bases, translation of the amino acid sequence was not possible. With the amino acid sequence available it would be possible to determine if the goldfish SRP54 gene codes for the same methionine rich domain as its mammalian counterpart.


Full Paper

Acknowledgments

The author would like to express sincere thanks to Sabrina Volpi, Rocco Coli, and Ira Daly, without whose support this project could not have been completed. The author also thanks Dr. Berish Y. Rubin for his help with developing the idea for the project and allowing the use of his lab.

References
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