Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 101 (19), 7375-80

RNA Surveillance Down-Regulates Expression of Nonfunctional Kappa Alleles and Detects Premature Termination Within the Last Kappa Exon

Affiliations

RNA Surveillance Down-Regulates Expression of Nonfunctional Kappa Alleles and Detects Premature Termination Within the Last Kappa Exon

Laurent Delpy et al. Proc Natl Acad Sci U S A.

Abstract

Random V(D)J junctions would generate nonfunctional and/or out-of-frame sequences in about two-thirds of cases and result in abundant transcripts encoding truncated proteins. Although allelic exclusion at the DNA recombination level ensures that a single allele is functional, the frequent biallelic rearrangements need additional mechanisms to down-regulate aberrant transcripts in those cells with both a functionally and a nonfunctionally rearranged allele. The process of nonsense-mediated decay targets aberrantly rearranged Ig heavy-chain transcripts, but the situation of light-chain mRNAs is more complex, because they do not meet the usual requirements for nonsense-mediated decay and most often lack a spliceable intron downstream of the premature termination. We studied immunoglobulin heavy-chain -/- pro-B cells in which light chain genes get rearranged and expressed in the absence of any selection for the assembly of a functional B cell receptor. Using this model, we show that the whole kappa locus is accessible in pro-B cells and allows the assembly of a broad spectrum of VkappaJkappa segments, most of which are out-of-frame. This model provides an evaluation of the in vivo efficiency of RNA surveillance toward aberrant kappa mRNAs produced in pro-B cells. Our data show that nonfunctional kappa transcripts are excluded from the mature mRNA pool not only by detecting termination in an upstream exon but also by detecting changes in the position of termination within the last exon. Similar mechanisms efficiently down-regulate nonfunctional kappa transcripts arising in normal mature B cells due to the biallelic transcription of rearranged kappa genes.

Figures

Fig. 1.
Fig. 1.
Vκ-Jκ junctions in bone marrow pro-B cells from N/N homozygous mice. Sequences are given with reference to the name of the corresponding germ-line Vκ and Jκ segment. Dashes indicate junctional deletions; N insertions (bold) and P insertions are indicated in between. Italicized nucleotides encode the CDR3 segment (whose length is indicated in parentheses); nonfunctional sequences are marked by an asterisk with indication of the +1 or +2 frameshift or of an in-frame stop codon (in upper case).
Fig. 2.
Fig. 2.
Schematic structure of the various classes of Vκ-Jκ rearrangements. Positions of the PTC resulting from a frameshifted rearrangement and of the normal in-frame stop codon are indicated (***).
Fig. 3.
Fig. 3.
CDR3 lengths and out-of-frame κ junctions. Bone marrow samples from either WT/WT or IgH-/- animals blocked at the pro-B cell stage were analyzed by high-resolution capillary electrophoresis. Data from a representative experiment are shown at the DNA level (A), the primary RNA level (B), and the mature RNA level [without cycloheximide (C) and with cycloheximide (D)]. Peaks corresponding to in-frame rearrangements are colored gray (including the dominant CDR3 with a canonical length of 27 nt); NF/F ratios are indicated. (E) Control of protein synthesis inhibition in cycloheximide-treated vs. untreated splenocyte cultures (metabolically labeled Ig were adsorbed on protein G and separated on a polyacrylamide gel).
Fig. 4.
Fig. 4.
Relative frequencies of out-of-frame κ junctions. NF/F ratios were estimated at the DNA, primary transcript, and mature mRNA levels by multiple experiments of capillary electrophoresis in IgH-/- pro-B cells or in tissues from normal mice (bone marrow, spleen, and κ-expressing sorted splenocytes). Filled diamonds represent values determined for Vκ-Jκ1 junctions in DNA and primary RNA and for Vκ-Jκ-Cκ junctions in mature transcripts; open squares represent Vκ-Jκ5 junctions in DNA and primary RNA.

Similar articles

See all similar articles

Cited by 12 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback