Elsevier

Molecular Brain Research

Volume 63, Issue 1, 10 December 1998, Pages 98-104
Molecular Brain Research

Research report
Characterization of transcript processing of the gene encoding precerebellin-1

https://doi.org/10.1016/S0169-328X(98)00264-2Get rights and content

Abstract

Precerebellin-1 (Cbln1) is a cerebellum-specific protein that shares significant sequence identity with the globular domains of the complement components C1qA, B and C, suggesting some common aspects of function and/or structure. As the C1q complex is composed of heterotrimers of C1qA, B and C it was hypothesized that multiple precerebellins may exist in a ternary complex. Northern blotting for cbln1 revealed multiple bands that could represent further family members or alternatively spliced variants. To discriminate these alternatives, probes derived from different regions of the cbln1 gene were used to identify and clone the transcripts detected on Northern blots. Four independent transcripts were repeatedly cloned from an adult mouse cerebellum cDNA library. Upon sequencing, all of these clones were found to be derived from the cbln1 gene and no additional precerebellin-related genes were isolated. Moreover, these clones accounted for the four cbln1-hybridizing bands (1.9, 2.2, 3.2 and 5.5 kb) detected on Northern blots of adult cerebellum RNA. With one possible exception, these clones were all derived through alterations in the 3′-untranslated region (3′-UTR) of cbln1 that did not affect the coding sequence. This was achieved by the use of two polyadenylation sites and alternative (non-canonical) splicing in the 3′-UTR. Some additional variation in mRNA structure is provided by the use of alternative transcription start sites in cbln1. The possible significance of this level of diversity in the 3′-UTR is discussed.

Introduction

A novel family of proteins, termed the precerebellins have been described that are expressed in the adult and developing nervous systems 8, 23, 24. The two known members of the precerebellins (Cbln1 and Cbln2) belong to a larger superfamily that includes a number of atypical collagens [23]. In particular, both precerebellins share significant amino acid homology with the globular (non-collagen) domains of the complement components C1qA, B and C 23, 24. This implies that these two subfamilies of proteins may share some common aspects of structure and/or function. The C1 complex comprises of two functional components: a recognition core made up of C1q proteins and an effector component comprising of the C1s and C1r proteases and the C1 inhibitor [10]. The recognition core consists of six heterotrimers of C1qA, B and C that bind to surface immunoglobulin. Since the globular domains of C1q contain the immunoglobulin-binding region, we speculated that the precerebellins might also assemble into a ternary complex that binds to an immunoglobulin superfamily member in the nervous system [23]. Furthermore, since there are three structurally related C1q subunits encoded by independent genes, we also supposed that one or more additional precerebellins might exit.

On Northern blots, probes to human cbln1 detected multiple mRNA species that could not be accounted for by cross-hybridization to cbln2 transcripts 8, 23. This raised the possibility that there are either additional members of the cbln gene family or that cbln1 undergoes alternative splicing. To distinguish these possibilities we have isolated and characterized cDNA clones that hybridize to a series of probes derived from various exons and introns of the cbln1 gene. No further cbln1-related genes were identified. Rather all of the mRNA species observed in Northern blot could be accounted for by the use of alternative polyadenylation sites and both conventional and atypical splicing of the 3′-UTR in cbln1 mRNA. Thus, several posttranscriptional mRNA processing mechanisms are used to increase the structural diversity of the 3′-UTR of cbln1 without changing the sequence of the protein itself. This surprising diversity of the 3′-UTR is discussed.

Section snippets

Genomic and cDNA clones for cbln1

A mouse genomic clone containing cbln1 was isolated previously as part of a genome mapping project [8]. This clone was sequenced by the dideoxy method as modified for a double stranded DNA template using a Sequenase 2.0 kit (USB). The general structure of this genomic clone, but not its sequence has been published previously [8].

cDNA clones for mouse cbln1 were obtained by screening an adult mouse cerebellum cDNA library [11]using standard techniques [12]. To obtain all cross-hybridizing

Results

Previously, as part of a gene mapping analysis we reported the isolation and general structure of a genomic clone encoding mouse cbln1 [8]. The sequence of relevant regions of this clone is depicted in Fig. 1. Based upon the precerebellin cDNA sequence it was possible to assign the initial intron-exon structure to this gene [[8]and Fig. 1, Fig. 2]. Exon 1 contains the relatively short 5′-untranslated region (5′-UTR) of precerebellin followed by an open reading frame (ORF) of 264 bp. The

Discussion

The cbln gene family presently comprises of two members, cbln1 and cbln2, that share a significant amino acid sequence homology with the globular (non-collagen) domain of the complement cascade components, C1qA, B and C 8, 23, 24. Since the globular domains in the C1q complex bind to the Fc region of membrane-associated immunoglobulin we have suggested that precerebellins may exist in homo- or heteromeric complexes that bind to brain-specific members of the immunoglobulin superfamily. Given the

Acknowledgements

This work was supported in part by NIH Cancer Center Support CORE Grant P30 CA21765 and by the American Lebanese Syrian Associated Charities (ALSAC).

References (25)

  • F.B. Gao

    Messenger RNAs in dendrites: localization, stability, and implications for neuronal function

    Bioessays

    (1998)
  • A. Gelot et al.

    Alpha-brain spectrin mRNA belongs to the population of intradendritically transported mRNAs

    NeuroReport

    (1996)
  • Cited by (0)

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