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Differential RNA expression and polyribosome loading of alternative transcripts of the Akap4 gene in murine
Rick W Nipper1, Vargheese Chennothukuzhi, Levent Tutuncu
1Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104, USA.
Abstract:
An X chromosome-linked gene, Akap4, is expressed only during spermiogenesis and encodes the major fibrous sheath protein of the mouse sperm flagellum. All sperm contain the AKAP4 protein even though only X chromosome-bearing spermatids express the gene, indicating that the Akap4 mRNA and/or protein must be shared among the conjoined spermatids via the intercellular bridges. There are two mouse Akap4 cDNA clones, Akap82 and Fsc1, which represent mRNAs that arise by alternative processing of a single gene. Although Akap82 and Fsc1 encode identical mature proteins, they differ in their 5' UTRs. We hypothesized that the expression pattern of these two mRNAs might be relevant to the issue of mRNA and/or protein transport into adjacent spermatids. Expression of both transcripts began in round spermatids, but the amount of the Akap82 transcript in condensing spermatids increased twofold relative to Fsc1. Significantly, only the Akap82 transcript was found on polyribosomes and translated in spermatids. These results indicate that the Akap82 transcript and/or its protein must be shared among the conjoined X and Y chromosome-bearing spermatids. Although Fsc1 was not polysomal, both the Akap82 and Fsc1 transcripts were deadenylated during spermiogenesis, suggesting that deadenylation is not always correlated with loading of mRNAs onto polyribosomes in germ cells. The distinct 5' UTR sequences in Akap82 and Fsc1 did not differ in their ability to regulate translation of reporter constructs either in vivo or in vitro. Antisense RNA transcripts complementary to both the Akap82 and Fsc1 mRNAs were present, suggesting that translatability may be regulated by these RNAs.
Insights
The Akap4 gene
Area of Science:
- Spermatogenesis and male reproductive biology
- Molecular and cellular biology
- Gene expression and regulation
Background:
- Akap4 is an X-linked gene crucial for sperm fibrous sheath formation.
- Akap4 expression occurs during spermiogenesis, but the protein is found in all sperm.
- Intercellular bridges likely facilitate sharing of Akap4 mRNA and protein between spermatids.
Purpose of the Study:
- To investigate the differential expression and regulation of two Akap4 mRNA variants (Akap82 and Fsc1).
- To determine the role of these variants in mRNA and protein transport between conjoined spermatids.
- To understand the mechanisms governing Akap4 translation during spermiogenesis.
Main Methods:
- Analysis of Akap4 transcript expression patterns in mouse spermatids.
- Detection of mRNA localization on polyribosomes to assess translation.
- In vivo and in vitro reporter assays to evaluate 5' UTR regulatory functions.
- Investigation of antisense RNA involvement in translational control.
Main Results:
- Both Akap82 and Fsc1 transcripts are expressed in round spermatids, with Akap82 increasing in condensing spermatids.
- Only the Akap82 transcript is found on polyribosomes and translated, suggesting its role in protein sharing.
- Deadenylation of both transcripts occurs during spermiogenesis, independent of polyribosome loading.
- Distinct 5' UTRs of Akap82 and Fsc1 do not differentially regulate translation in reporter assays.
- Antisense RNAs are present, indicating a potential regulatory mechanism for Akap4 translatability.
Conclusions:
- The Akap82 transcript is preferentially translated and likely responsible for Akap4 protein sharing in spermatids.
- Akap4 mRNA and protein transport via intercellular bridges ensures complete fibrous sheath formation.
- Deadenylation is not a strict prerequisite for mRNA loading onto polyribosomes in germ cells.
- Translational regulation of Akap4 may involve antisense RNAs, independent of 5' UTR sequence differences.
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