SHORT COMMUNICATION

Alternatively Spliced Variants of Gamma-Subunit of Muscle-TypeAcetylcholine Receptor in Fetal and Adult Skeletal Muscleof Mouse

Shafquat Azim • Abdul Rouf Banday •

Tarique Sarwar • Mohammad Tabish

Received: 18 October 2011 / Accepted: 23 March 2012 / Published online: 10 April 2012

� Springer Science+Business Media, LLC 2012

Abstract Gamma-subunit of nicotinic acetylcholine

receptor is encoded by chrng gene of mouse. This gene is

located on chromosome 1, spans 6.5 kb, and contains 12

exons and 11 introns. Previous studies have reported three

transcript variants (C1-3) produced by alternative splicing;

C1 contains all the 12 reported exons, C2 uses an in-frame

alternate splice site in exon-2, and C3 produced by exon-5

skipping. These variants differ in their channel kinetics and

opening times. In our study, we report the presence of two

new transcript variants (T1 and T2) of chrng expressed in

mouse postnatal day 3 and adult skeletal muscles. These

transcripts contain novel first coding exon either N1 or N2.

N1 is located in the 50 UTR, while N2 is an extended exon-2.

50 extension of exon-2 contains an initiation codon which

produces a novel transcript variant. Either of the two exons

can splice with the internal exons to produce mature tran-

scripts making different 50 ends of the transcripts. Conse-

quently, the proteins encoded by these two transcripts

differ at N-termini. The presence of N2 exon containing

transcript was further supported by the availability of EST

from the database. These new variants display heteroge-

neous properties. They differ in the presence of sig-

nal peptide, phosphorylation, and acetylation of their

amino acid residues of the new N-termini of the gamma

subunit.

Keywords Nicotinic acetylcholine receptor �Gamma subunit � Transcript variants � Isoforms

Introduction

The chrng gene encodes the gamma subunit (c-subunit) of

the muscle-type nAChR complex, and is largely expressed

in fetal muscle (Albuquerque et al. 2009). The c-subunit of

nAChR plays an important role in neuromuscular devel-

opment, neuromuscular signal transduction, neuromuscular

organogenesis, ligand binding, and insertion of receptors in

the membrane (McArdle et al. 2008; Kapur et al. 2006).

The c-subunit may be involved in interacting with rapsyn, a

cytoplasmic protein required for receptor clustering (Liu

et al. 2008). nAChRs are now important therapeutic targets

for various diseases, including myasthenia gravis, multiple

pterygium syndrome, Alzheimer’s and Parkinson’s disease,

and schizophrenia, as well as for the cessation of smoking

(Kalamida et al. 2007; Hoffmann et al. 2006).

Alternatively spliced variants of c-subunit have been

reported in rodents which increase the functional diversity

of AChR. In case of rats, alternative splicing of c-subunit

produces four isoforms (c1–c4) of the subunit (Villarroel

1999). c1 is a full length receptor subunit, while c2 and c4

are soluble proteins which may serve as a retrograde signal

reporting successful synaptic contacts. The isoform c3 is a

complete subunit which could contribute to the AChR

channel diversity observed in muscle (Villarroel 1999;

Villarroel and Sakmann 1996). In mouse, three transcript

variants C1, C2 and C3 have been reported earlier from

embryonic developmental stages of mouse which differ in

their 50 coding exons and the presence or absence of exon-5

(Mileo et al. 1995; Mural et al. 2002). These three variants

differ in their channel kinetics and channel opening times.

They increase the diversity of the receptors. Alternative

splicing plays a very important role in modulating the

receptor; therefore, we decided to study the transcript

variants of chrng gene in mouse. We identified two new

S. Azim � A. R. Banday � T. Sarwar � M. Tabish (&)

Department of Biochemistry, Faculty of Life Sciences,

A.M. University, Aligarh 202002, U.P., India

e-mail: tabish.bcmlab@gmail.com

123

Cell Mol Neurobiol (2012) 32:957–963

DOI 10.1007/s10571-012-9838-y

coding exons designated N1 and N2, either of which can

alternatively splice to internal exons producing two dif-

ferent transcript variants T1 and T2, respectively, from P3

(postnatal day 3) mouse skeletal muscle along with the

earlier published variants. However, C3 and T1 transcripts

were also observed in adult skeletal muscle. These tran-

scripts differ in their first coding exons. Their properties

appear to be highly heterogeneous. The differences in their

properties might serve for differential localization as well

as altered kinetics of the receptor.

Materials and Methods

Materials

Mice (A/J) were purchased from the Jamia Hamdard

University, New Delhi, India and bred in house. All ani-

mals were housed according to the Institutional Animal

Care and Use Committee and Guidelines. The total RNA

extraction kit was purchased from iNtRON Biotechnology,

Inc. (Gyeonggi-do, Korea). M-MuLV-Reverse Transcrip-

tase, High Fidelity PCR kit and nucleotide mix were pur-

chased from Fermentas (USA). 100 bp PCR DNA ladders

were purchased from GenScript (USA). The TOPO-TA

cloning kit II was obtained from Invitrogen Corp. (Carls-

bad, CA, USA). The Plasmid DNA miniprep kit and Qia-

quick PCR gel purification kits were purchased from

Qiagen, Inc. (Santa Clarita, CA, USA). Primers were cus-

tom synthesized from MWG Biotech, Pvt. Ltd., India. All

other chemicals used in the experiments were of molecular

biology grade.

RNA Preparation

Animals were euthanized with carbon dioxide. In case of

adults, the experiment was repeated using both male and

female mouse in independent experiments. 50 mg of the

skeletal muscle was excised from the thigh region of P3

(Postnatal 3 days, gender unknown) and adult (15 days old,

both male and female) mouse. The experiment was repe-

ated several times and each time the same age mouse was

used. Total cellular RNA of P3 and adult mouse skeletal

muscle was prepared using RNA Extraction Kit according

to the manufacturer’s instructions. The integrity of eluted

RNA was checked by denaturing agarose gel electropho-

resis and ethidium bromide staining.

Primers

Genomic sequence of chrng gene was downloaded from

NCBI with accession number GenBank ID NM009604 and

primers were designed. The sequence of the primers and

the expected sizes of the products are given below. The

direction and relative position of the primers are indicated

in Fig. 1a.

50 Rapid Amplification of cDNA Ends (50 RACE)

In order to investigate the presence of possible new splice

variants of the chrng gene, RACE was performed with 2 lg

of total RNA isolated from P3 and adult skeletal muscles

using a 50 RACE kit. The total RNA was annealed with

chrng specific exon-6 reverse primer (MREV2CHRNG:

50-CTC CGG GTC AAT GAA GAT CCA CTC AAT G)

according to the manufacturer’s instructions. The RACE

product was fractionated by electrophoresis using 1.2 %

agarose gel. Several bands were excised from the gel,

purified and sub-cloned into TOPO vector.

Reverse Transcriptase (RT)-PCR and Semi-Nested

PCR

50 RACE data were further confirmed by RT-PCR. First

strand cDNA was synthesized from 2 lg of total RNA

primed with gene-specific primer MREV1CHRNG

(50-CTT GCG CTG GAT AAG CAG GTA GAA CAC)

designed from 7th exon using Reverse Transcriptase at

43 �C for an hour in a total volume of 20 ll. 5 ll of the

single-stranded cDNA was then amplified by touchdown

PCR using MREV2CHRNG and 1st exon specific primers

as downstream and upstream primers, respectively, and

PCR kit in 50 ll of reaction mixture. PCR was performed

as follows: denaturation at 94 �C for 4 min, 1 cycle; 30

cycles were repeated at 93 �C for 30 s; 66 �C for 30 s with

a decrease of 0.3 �C per cycle; and 72 �C for 45 s. Final

extension at 72 �C was done for 8 min. Final product was

subjected to electrophoresis on a 1.2 % agarose gel, and

photographed on a UV transilluminator. A summary of

primer sequences, amplicons and their product size are

summarized below:

Transcript Name: exon specific

forward primer

Exons in

amplicon

Semi-nested

PCR product

size (bp)

C1 MCTRCHRNG: 50-CAG

AAC TGA GGC ACC

ATG CAA GG

E1–E2–E3–

E4–E5–E6

609

C2 MCTRCHRNG: 50-CAG

AAC TGA GGC ACC

ATG CAA GG

E1–E200–E3–E4–

E5–E6

546

C3 MCTRCHRNG: 50-CAG

AAC TGA GGC ACC

ATG CAA GG

E1–E2–E3–

E4–E6

453

958 Cell Mol Neurobiol (2012) 32:957–963

123

Table a continued

Transcript Name: exon specific

forward primer

Exons in

amplicon

Semi-nested

PCR product

size (bp)

T1 MN1FCHRNG: 50-CAC

GGA TAC ACA CTG

GCC AGA ATG TTG

N1–

E2 ? E20–E3–E4–E6

787

T2 MN2FCHRNG: 50-CAA

GAT TAC AGT GGA

TGG AGG GTC TGG

N2E2–E3–

E4–E6

674

Subcloning and Sequencing of RT-PCR Products

50 RACE and RT-PCR amplified products were electro-

phoresed on 1.2 % (w/v) agarose gels. Anticipated bands

were excised from the gels and DNA was purified using

PCR gel purification kit. The purified DNA was sub-

cloned in TOPO vector. After transformation, E. coli

JM109 were grown overnight at 37 �C and plasmid DNA

was purified. Plasmids containing the inserts were

sequenced using either M13 forward or reverse primer

(Sanger et al. 1977).

Database Analysis

Homology and similarity searches of the obtained nucleo-

tide sequences were performed by means of the BLASTN

non-redundant database (http://www.ncbi.nlm.nih.gov/

BLAST). Alignment analysis was carried out using the

Gene stream Align tool (http://www2.igh.cnrs.fr/bin/

align-guess.cgi) and ClustalW tool available at www.ebi.

ac.uk/clustalw (Altschul et al. 1997). ExPASy tools at

http://ca.expasy.org/ were used to predict the MW/pI and

post-translational modifications in amino acid sequences.

Results and Discussion

Alternatively Spliced Two Novel Transcripts of chrng

Having Different 50 Exons

In mouse, the chrng gene encoding gamma subunit of

acetylcholine receptor is located on chromosome 1, spans

6.5 kb containing 12 exons and 11 introns. cDNA corre-

sponding to the chrng transcript is present in the database

with the accession number NM009604 and it codes for a

protein containing 519 amino acids. Three transcript

Fig. 1 Exon–intron organization of chrng gene and the transcript

variants; exons and introns are presented by rectangles and intercon-

necting straight lines, respectively. Exons as part of transcript is

shown with filled rectangles and empty rectangles represent the exon/

part of exon skipped from the transcript. Splicing patterns are shown

by bent lines between the exons. a Genomic DNA showing 12 exons

and 11 introns in the gene. b Three different transcripts (C1, C2, and

C3) that arise due to splicing and has been published earlier. C1

transcript contains all exons, C2 contains all exons but slightly

shortened exon-2 (E200), and C3 contains all exons except exon-5.

c Newly identified two transcripts N1 and N2. Transcript N1 has a

new 1st exon located upstream of the exon E1 that splices with exon-2

extended toward 30 end. N2 transcript contains 50 end extended exon-

2 having internal initiation codon. Primer positions and directions are

indicated by arrows above the exons. Exons and introns are not to

scale. d Agarose gel electrophoresis of 50 RACE product from P3 and

adult skeletal muscle. Amplified 50 RACE product was electropho-

resed on 1.2 % gel and stained with ethidium bromide. M stands for

100 bp DNA ladder; ?RT P3 ?RT adult for RACE products in the

presence of RT from P3 and adult skeletal muscle, respectively; and

-RT P3 -RT adult are RACE products in absence of RT from P3 and

adult skeletal muscle, respectively

Cell Mol Neurobiol (2012) 32:957–963 959

123

variants have been reported earlier (Fig. 1a, b), C1 is the

largest transcript encoding full length cl, C2 transcript

(CRA_a) uses an in-frame alternate splice site in exon-2,

causing deletion of 63 nucleotides from 50 end of exon-2

(E200), and C3 transcript results due to the skipping of exon-5

encoding cs (Mileo et al. 1995; Mural et al. 2002).

In order to study the 50 alternatively spliced transcript

variants, 50 RACE was performed on the RNA isolated

from P3 and adult skeletal muscle. The products when

fractionated by agarose gel electrophoresis revealed several

bands as shown in Fig. 1d. These bands were excised from

the gel; DNA was purified, subcloned, and sequenced.

Comparison of sequences obtained identified the already

published sequences (Fig. 1a, b) corresponding to C1, C2,

and C3 transcripts in addition to two new transcript vari-

ants T1 and T2 (Fig. 1c). Further analysis of the two new

sequences of the transcripts with respect to the genomic

sequence of chrng gene revealed that these new sequences

were part of chrng gene and they differed in their first

coding exon. Two new exons were identified as N1 and N2.

Exon-N1 is located 1132-bp upstream of the previously

reported 1st exon (E1). N1 splices with exon-2, which has

an extended 30 end that comes from a portion of the 2nd

intron and forms a new second exon designated as

E2 ? E20. The in frame-usage of an alternate donor site at

the 30 end of exon-2 introduces a stretch of 189 nucleotides

in the published exon-2. The splicing pattern of the new

second exon-E2 ? E20 is conserved which splices with

published exon-3 to produce T1 transcript. The other newly

identified exon-N2 is an extended form of published exon-2

at the 50 end. An initiation codon is present in the extended

part of exon-2. N2 splices with published exon-3 to pro-

duce a new transcript variant T2. The splicing patterns of

different exons are depicted in Fig. 1b, c.

The 50 RACE results were confirmed by RT-PCR fol-

lowed by semi-nested PCR. 50 RACE sequencing results

were used for designing the primers specific to the new

exons N1 and N2. The semi-nested PCR products were

electrophoresed; DNA bands were excised, subcloned, and

sequenced. The agarose gel electrophoresis of semi-nested

PCR products showed bands corresponding to the expected

sizes of all the published transcripts as well as T1 and T2

transcripts (Fig. 2a) in P3 skeletal muscle. However, only

C3 and T1 transcripts were amplified from adult skeletal

muscle (Fig. 2b). The sequences of these new transcripts

were submitted in the GenBank and were assigned the

accession numbers JN164665 and JN164666 for N1 and

N2, respectively. Aligning the translated amino acid

sequences of these new transcripts with that of the already

published sequences by web based program ‘ClustalW’ is

shown in Fig. 2c revealed that these sequences were dif-

ferent only in their N-terminal regions, while the rest of the

sequences from exon-3 onward were identical. Expressed

sequence tag (EST) searches by the nucleotide sequence of

exon-N1 found no hit in the database; however, exon-N2

was found to hit several sequences from the database

with the accession numbers CJ177240, CJ185052, and

CJ176146. All these ESTs were confined to 14.5 days

mouse embryo and in Rathke’s pouches. Thus, the presence

of T2 transcript was limited to the early stages of devel-

opment which is also supported by EST.

In Silico Analysis of the Amino Acid Sequences

Encoded by the New Transcripts

In order to understand the function of different isoforms of

CHRNG, we performed comparative post-translational

studies in silico using conceptual translated sequences

differing at the N-termini of the protein encoded by 1st and

2nd exons of the transcripts depicted in Table 1. Interest-

ingly, we observed the presence of a cleavable signal

peptide responsible for differential targeting in the pub-

lished isoforms as well as isoform encoded by N2 tran-

script. However, such sequence is absent in N1 encoding

CHRNG isoform. Absence of signal peptide in N1 encoded

isoform might play a role in negative regulation of the

receptor similar to the rat thyrotropin receptor, where

deletion of a sequence containing the putative signal pep-

tide formed non-functional receptors (Akamizu et al.

1990). The conserved residues K 34, S 111 and F 172 (Sine

et al. 1995) are found to be preserved in these forms. Both

the newly identified variants T1 and T2 have two potential

O-glycosylation sites whereas C1, C2, and T1 have puta-

tive N-glycosylation site. C1and C2 has two, T1 has ten

and T2 has five potential phosphorylation sites. C1, C2, and

T2 have Thr residue, which can be potentially phosphor-

ylated by PKA enzyme; however, T1 has a Ser residue

which can be phosphorylated by PKC. Green et al. (1991)

reported that the phosphorylation of unassembled c-subunit

in Torpedo increases the efficiency of assembly of all the

four subunits of nAChR. However, Jayawickreme et al.

(1994) reported that the presence of c-subunit increases the

efficiency of assembly of nAChR, but phosphorylation of

the subunit is not found to play any role. This indicates a

discrepancy between two different groups of investigators.

However, further investigation is required to understand

the role of phosphorylation in assembly of nAChR. C1 and

C2 as well as N1 and N2 contain G3 residue which can be

potentially acetylated; however, N1 contains an additional

acetylation site T2. Although majority of eukaryotic pro-

teins are N-terminally acetylated (Nt-acetylated), the

function of this modification is largely unknown (Hwang

et al. 2010; Mogk and Bukau 2010). For some proteins

such as actin, Nt-acetylation has been reported to affect

960 Cell Mol Neurobiol (2012) 32:957–963

123

protein functionality, e.g., non-acetylated actin is less

efficient in assembling microfilaments (Polevoda et al.

2003). Recently, the other role suggested for acetylation is

that Nt-acetylation of a protein can function as a degra-

dation signal playing a role in protein turnover and

homeostasis (Hwang et al. 2010).

Fig. 2 a Agarose gel electrophoresis of the RT-PCR products from

P3 mouse muscle. 1 DNA marker 100 bp ladder. 2 RT-PCR products

obtained using primers from the published sequence. It contains three

bands each of which corresponded to the expected size of C1

(609 bp), C2 (546 bp), and C3 (453 bp). 3 Transcript variant T1 with

anticipated size of 787 bp. 4 Transcript variant T2 with anticipated

size of 674 bp. b Agarose gel electrophoresis of the RT-PCR products

from adult mouse muscle. 1 DNA marker 100 bp ladder. 2 RT-PCR

product for C3 with anticipated size of 453 bp. 3 Transcript variant

T1 with anticipated size of 787 bp. 4 A very faint band of transcript

variant T2 with anticipated size of 674 bp. c Multiple sequence

alignment (ClustalW) of the deduced amino acid sequences of the

published transcripts (C1, C2, and C3) and the newly identified

transcripts (T1 and T2). Asterisks sign indicates the identical residues

present in all five sequences. All sequences are identical from third

exon onward. Accession numbers are given at the end of each

sequence

Cell Mol Neurobiol (2012) 32:957–963 961

123

The diversity of the receptors is increased through

alternative splicing of the different subunits of the nAChRs.

In addition to the previously reported transcript variants,

our studies report the presence of two new transcript vari-

ants of chrng gene produced through the usage of alternate

50 exons in P3 skeletal muscle and a new variant in adult

skeletal muscle. In silico analysis revealed heterogeneous

nature of these transcripts. The study of these variants is

important to fully understand the functioning, localization,

properties and difference in expression observed in fetal and

adult muscle of the muscle-type nAChRs, and the under-

lying causes of various disorders involving nAChRs.

Acknowledgments The authors are thankful to the DBT and CSIR

New Delhi, India for generous funding and Aligarh Muslim Univer-

sity for providing necessary facilities.

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Table 1 In silico analysis of the translated amino acid sequence of first two exons in published and new transcripts

Predictions E1 ? E2 (C1) E1 ? E200 (C2) N1 ? E20 (T1) N2 ? E2 (T2)

Amino acid

sequence

MQGGQRPQLLLLL

LAVCLGAQSRNQE

ERLLADLMRNYDP

HLRPAERDSDVVN

VSLKLTLTNLISL

MQGGQRPHLLLLLLA

VCLGSGLRPAERDSNV

VNVSLKLTLTNLISL

MTGGPRAQSRNQEERLLAD

LMRNYDPHLRPAERDSDVV

NVSLKLTLTNLISLVSNRRR

GMMDITQGHRLAGEINEC

WGSNPSTRNSRGSYNGVGR

THETPGATTRCEAEGLLVL

MEGLGVTCTLPSPSTLPAP

SLHLIPLCLLLLWSEPPPS

VAGAQSRNQEERLLADL

MRNYDPHLRPAERDSDV

VNVSLKLTLTNLISL

No. of amino acid

residues

65 46 115 87

Molecular weight 7311.51 4924.88 12759.33 9497.03

Isoelectric point 6.53 9.49 8.84 5.11

Net-O-

glycosylation

No No T2, T96 T7, T9

Net-N-

glycosylation

51NVS 33NVS 39NVS No

Net phos. S: 2, T: 0, Y: 0

S22, S54

S: 2, T: 0, Y: 0

S29, S35

S: 6, T: 3, Y: 1

S9, S35, S41, S82, S86, S89, T99,

T103, T104, Y90

S: 5, T: 0, Y:0

S12, S14, S38, S70, S76

Net phosK PKA at T58 PKA at T39 PKC at S54 PKA at T80

Signal peptide Contains signal peptide

AQS–RN

Contains signal peptide

GSG–LR

Non-secretory protein Contains signal peptide

VAG–AQ

Acetylation G3 G3 T2 and G3 G3

A comparative analysis of the amino acid sequences suggesting a high percent of heterogeneity in the variants encoded by chrng gene. The

superscript donates the position of the amino acid undergoing modification

962 Cell Mol Neurobiol (2012) 32:957–963

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