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A novel isoform of human membrane cofactor protein (CD46) mRNA generated by intron retention
A J Pollard1, B F Flanagan, D J Newton
1Department of Immunology, University of Liverpool, UK.
Abstract:
The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine-threonine-rich region (ST-A), together with an antisense exon 12 primer, RT-PCR with primers for other exons both 5' and 3' of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5' and 3' to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.
Insights
Researchers discovered a novel, truncated membrane cofactor protein (MCP; CD46) isoform in human immune cells. This isoform results from intron retention, potentially affecting protein function and cellular localization.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Membrane cofactor protein (MCP; CD46) is a key regulator of the complement system.
- MCP plays a crucial role in immune cell function and immune response modulation.
- Understanding MCP mRNA expression is vital for comprehending complement regulation.
Purpose of the Study:
- To investigate MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC).
- To identify and characterize novel MCP mRNA transcripts and their encoded protein isoforms.
- To elucidate the molecular mechanisms underlying alternative splicing or intron retention in MCP gene expression.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) using primers specific for all 14 exons of human MCP.
- RT-PCR analysis with primers flanking exon 7 and across introns 6 and 7.
- Genomic DNA and cDNA comparison via PCR and sequence analysis.
- Southern and Northern blotting utilizing an intron 6-specific probe.
Main Results:
- A novel, larger-than-predicted MCP mRNA transcript was consistently detected in PBMC.
- This transcript contains additional sequences 5' and 3' to exon 7, including the complete sequences of intron 6 and intron 7.
- Confirmation of intron 6 retention in a 4.8-kb mRNA transcript in human PBMC.
- Sequence analysis revealed a stop codon within intron 6, leading to a truncated MCP isoform (MCPi).
Conclusions:
- A novel MCP isoform (MCPi) is generated through intron 6 retention in human PBMC.
- MCPi contains the four invariant short consensus repeat (SCR) regions and a unique C-terminal tail.
- The unique C-terminal region of MCPi may promote endoplasmic reticulum retention, suggesting altered protein localization and function.