Occurrence and Sequence Complexity of Polyadenylated RNA in Squid Axoplasm

Abstract

Axoplasmic RNA from the giant axon of the squid (Loligo pealii) comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/microgram of total RNA) suggests that the poly(A)+ RNA population makes up approximately 0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments greater than 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower-reacting component (moderately abundant and rare sequences). The latter component accounts for approximately 56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of approximately 2.7 kilobases and 3.1 X 10(2) kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents approximately 0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

“…The second argument is that many components of translation machinery are found in axons, including in squid giant axoplasm, and therefore this serves as prima facie evidence that translation must be occurring in this cellular compartment (Alvarez et al 2000;Giuditta et al 2008). These data include reports of abundant tRNAs (Black and Lasek 1977;Ingoglia et al 1983), mRNAs (Capano et al 1987;Gioio et al 1994;Kaplan et al 1992;Rapallino et al 1988), various elongation and initiation factors (Giuditta et al 1980;Giuditta et al 1986;Giuditta et al 1991;Kar et al 2013), rRNA (Giuditta et al 1980;Perrone-Capano et al 1999), and even polyribosomes that can be translated in vitro to produce various proteins (Giuditta et al 1991;Sotelo et al 1999). Given this apparent translation capacity of squid axoplasm it should then be possible for isolated axoplasm to synthesize proteins directly from radioactive amino acids.…”

Local Gene Expression in Axons and Nerve Endings: The Glia-Neuron Unit

“…These data were confirmed and extended by kinetic hybridization assays of polyadenylated axoplasmic RNA with its cDNA. This raised the estimated number of axoplasmic mRNAs to at least 200 different species (256). Such a high number was remarkable for an axon, even if kinetic hybridization analyses indicated that polyadenylated RNA from the axon cell bodies displayed much greater sequence complexity, comprising several thousand mRNAs.…”

Differential Transport and Local Translation of Cytoskeletal, Injury-Response, and Neurodegeneration Protein mRNAs in Axons

“…Localized protein synthesis in dendrites is a well-accepted mechanism that that can be regulated by neurotransmitters and trophic factors (Steward, 2002). Evidence for protein synthesis in axons was initially provided from studies in invertebrates (Capano et al, 1987;Giuditta et al, 1991;Davis et al, 1992;Chun et al, 1996;van Minnen et al, 1997). Subsequent work showed that developing vertebrate axons contain mRNAs and actively synthesize proteins (Olink-Coux and Hollenbeck, 1996;Bassell et al, 1998;Eng et al, 1999;Zhang et al, 1999;Aronov et al, 2001;Campbell and Holt, 2001; Lee and Hollenbeck, 2003).…”