Heavy Chain Primary Transcripts Undergo Differential RNA Processing

Processing of an immunoglobulin heavy-chain primary transcript can yield several different mRNAs, which explains how a single B cell can produce secreted or membrane-bound forms of a particular immunoglobulin and simultaneously express IgM and IgD.

EXPRESSION OF MEMBRANE OR SECRETED IMMUNOGLOBULIN

As explained in Chapter 4, a particular immunoglobulin can exist in either membrane-bound or secreted form. The two forms differ in the amino acid sequence of the heavy-chain carboxyl-terminal domains (CH3/CH3 in IgA, IgD, and IgG and CH4/CH4 in IgE and IgM). The secreted form has a hy-drophilic sequence of about 20 amino acids in the carboxyl-terminal domain; this is replaced in the membrane-bound form with a sequence of about 40 amino acids containing a hydrophilic segment that extends outside the cell, a hy-drophobic transmembrane segment, and a short hydrophilic segment at the carboxyl terminus that extends into the cytoplasm (Figure 5-16a). For some time, the existence of these two forms seemed inconsistent with the structure of germline heavy-chain DNA, which had been shown to contain a single CH gene segment corresponding to each class and subclass.

The resolution of this puzzle came from DNA sequencing of the C. gene segment, which consists of four exons (C.1, C.2, C.3, and C.4) corresponding to the four domains of the IgM molecule. The C.4 exon contains a nucleotide sequence (called S) at its 3' end that encodes the hydrophilic sequence in the CH4 domain of secreted IgM. Two additional exons called M1 and M2 are located just 1.8 kb downstream from the 3' end of the C..4 exon. The M1 exon encodes the transmembrane segment, and M2 encodes the cytoplasmic segment of the CH4 domain in membrane-bound IgM. Later DNA sequencing revealed that all the CH gene segments have two additional downstream M1 and M2 exons that encode the transmembrane and cytoplasmic segments.

The primary transcript produced by transcription of a rearranged . heavy-chain gene contains two polyadenylation signal sequences, or poly-A sites, in the C. segment. Site 1 is located at the 3' end of the C..4 exon, and site 2 is at the 3' end of the M2 exon (Figure 5-16b). If cleavage of the primary transcript and addition of the poly-A tail occurs at site 1, the M1 and M2 exons are lost. Excision of the introns and splicing of the remaining exons then produces mRNA encoding the secreted form of the heavy chain. If cleavage and polyadenylation of the primary transcript occurs instead at site 2, then a different pattern of splicing results. In this case, splicing removes the S sequence at the 3' end of the C. 4 exon, which encodes the hydrophilic carboxyl-terminal end of the secreted form, and joins the remainder of the C. 4 exon with the M1 and M2 exons, producing mRNA for the membrane form of the heavy chain.

Thus, differential processing of a common primary transcript determines whether the secreted or membrane form of an immunoglobulin will be produced. As noted previously, mature naive B cells produce only membrane-bound antibody, whereas differentiated plasma cells produce secreted antibodies. It remains to be determined precisely how naive B cells and plasma cells direct RNA processing preferentially toward the production of mRNA encoding one form or the other.

SIMULTANEOUS EXPRESSION OF IgM AND IgD

Differential RNA processing also underlies the simultaneous expression of membrane-bound IgM and IgD by mature B cells. As mentioned already, transcription of rearranged heavy-chain genes in mature B cells produces primary transcripts containing both the C. and C8 gene segments. The C. and C8, gene segments are close together in the rearranged gene (only about 5 kb apart), and the lack of a switch site between them permits the entire VDJC.C8 region to be transcribed into a single primary RNA transcript about 15 kb long, which contains four poly-A sites (Figure 5-17a). Sites 1 and 2 are associated with C., as described in the previous section; sites 3 and 4 are located at similar places in the C8 gene segment. If the heavy-chain transcript is cleaved and polyadenylated at site 2 after the C. exons, then the mRNA will encode the membrane form of the . heavy chain (Figure 5-17b); if polyadenylation is instead further downstream at site 4, after the C8 exons, then RNA splicing will remove the intervening C. exons and produce mRNA encoding the membrane form of the 8 heavy chain (Figure 5-17c).

Since the mature B cell expresses both IgM and IgD on its membrane, both processing pathways must occur simultaneously. Likewise, cleavage and polyadenylation of the primary heavy-chain transcript at poly-A site 1 or 3 in

556-

SS bridge

COOH

SS bridge

COOH

Key:

Hydrophilic Hydrophobic

556-

Encoded

556 Outside 568

Encoded by M1 and M2 ^ exons of C|

Membrane

594597 Cytoplasm

556 Outside 568

Membrane

594597 Cytoplasm

COOH

Secreted |

Membrane |

Primary H-chain —|-transcript

Site 1

Poly-A Poly-A site 1 site 2

Polyadenylation Site 2

RNA transcript for secreted | L V DJ J |1 |2 |3 |4 S

mRNA encoding secreted | chain

FIGURE 5-16

Expression of secreted and membrane forms of the heavy chain by alternative RNA processing. (a) Amino acid sequence of the carboxyl-terminal end of secreted and membrane ^ heavy chains. Residues are indicated by the single-letter amino acid code. Hydrophilic and hydrophobic residues and regions are indicated by purple and orange, respectively, and charged amino acids are indicated with a + or —. The white regions of the

Poly-A Poly-A site 3 site 4

RNA transcript for membrane | L V DJ J |2 |3 |4 S M1 M2

RNA transcript for membrane | L V DJ J |2 |3 |4 S M1 M2

<A)n mRNA encoding membrane | chain sequences are identical in both forms. (b) Structure of the primary transcript of a rearranged heavy-chain gene showing the C^ exons and poly-A sites. Polyadenylation of the primary transcript at either site 1 or site 2 and subsequent splicing (indicated by V-shaped lines) generates mRNAs encoding either secreted or membrane ^ chains.

(a) H-chain primary transcript

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  • Dylan Kelly
    Is polyadenylation site hydrophobic or hydrophilic?
    8 years ago

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