Worm Breeder's Gazette 11(2): 75
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
A subset of C. elegans mRNAs receives a 22 nucleotide leader from a precursor RNA called SL RNA. This RNA has the hallmarks of a typical snRNA: it is bound to the 'Sm' proteins and it has the unusual cap nucleoside, trimethylguanosine (TMG). Because this cap is on the 5' end of SL RNA it is transferred to recipient RNAs, along with the 22 nucleotides, during trans-splicing. Originally we believed this unusual cap was subsequently altered or removed, but it isn't. Since translation in eukaryotes is initiated by binding of an initiation factor, eIF-4E, to the cap (normally monomethylguanosine) the presence of a different cap on some mRNAs could have important functional consequences. For this reason (and also because a reviewer suggested it might be a good idea) we undertook to find out whether these TMG-capped mRNAs are found on polysomes. We made polysomes from young adult worms by a procedure adapted from a method for making plant polysomes. The procedure is briefly outlined below. We then used anti-TMG antibodies to demonstrate that actin-1 and actin-3 mRNAs found in the polysome preparation are indeed TMG capped. We conclude that TMG-capped mRNAs are translated in C. elegans. Several mechanisms by which translation may be initiated on these mRNAs are possible: 1. C. elegans eIF-4E might recognize both kinds of cap structure. 2. C. elegans may contain a special initiation factor capable of recognizing TMG. 3. TMG-capped mRNAs might initiate by a cap-independent mechanism, perhaps by recognition of the SL sequence. The Polysome Prep Worms ( ~105/sucrose gradient) are sucrose floated, washed in M9, frozen in liquid N2 and ground to a fine powder in a pre-chilled mortar with liquid N2. The worm powder is resuspended in 3 volumes of polysome buffer (0.2M Tris.HCl, pH 8.5; 35 mM MgCl2; 0.2 M KCl; 5 mM DTT; 15 mM EGTA; 0.5% NP-40). Following a brief 4 C, 4000xG spin to remove debris, the supernatant is layered on a 20-50% (W/W) sucrose gradient in the same buffer. The gradients are spun at 25K at 4 C 2 hr in a SW27 rotor. A control in which 100 mM EDTA is added to all buffers is routinely included. O.D.254 profiles show a broad diffuse peak in the region where polysomes are expected and only in the absence of EDTA. The broad peak is rich in mRNAs as anticipated.