Poly + mRNA

FIGURE 4.50 FVjrifIcation of mRNA on Toyopearl HW-65F. Column Toyopearl HW-65F. 25 mm X 90 cm. Sample 40 mg of poly(A) RNA/5 ml of formamide, sample from silkworm. Elution 20 mA1 sodium citrate, 5 mM EDTA, 0.5% SDS, 6 M urea, pH 3.5. Flow rate 24 ml/hr. Detection UV at 254, total mRNA activity (—), mRNA activity for major plasma proteins (------------). 

In one of the early experiments designed to elucidate the genetic code, Marshall Nirenberg of the U.S. National Institutes of Health (Nobel Prize in physiology or medicine, 1968) prepared a synthetic mRNA in which all the bases were uracil. He added this poly(U) to a cell-free system containing all the necessary materials for protein biosynthesis. A polymer of a single amino acid was obtained. What amino acid was polymerized ... 

The eukaryotic expression cassette is the part of an expression vector that enables production of a protein in a eukaryotic cell. The cassette consists of a eukaryotic promoter for mRNA transcription, the gene and an mRNA termination and processing signal (Poly-A signal). 

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

Small nuclear RNAs (snRNAs), a subset of these RNAs, are significantly involved in mRNA processing and gene regulation. Of the several snRNAs, Ul, U2, U4, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA (Chapter 37). The U7 snRNA may be involved in production of the correct 3 ends of histone mRNA—which lacks a poly(A) tail. The U4 and U6 snRNAs may also be required for poly(A) processing. 

The relationship between hnRNA and the corresponding mature mRNA in eukaryotic cells is now apparent. The hnRNA molecules are the primary transcripts plus their early processed products, which, after the addition of caps and poly(A) tails and removal of the portion corresponding to the introns, are transported to the cytoplasm as mature mRNA molecules. 

As mentioned above, mammahan mRNA molecules contain a 7-methylguanosine cap structure at their 5 terminal, and most have a poly(A) tail at the 3 terminal. The cap stmcmre is added to the 5 end of the newly transcribed mRNA precursor in the nucleus prior to transport of the mELNA molecule to the cytoplasm. The S cap of the RNA transcript is required both for efficient translation initiation and protection of the S end of mRNA from attack by S —> S exonucleases. The secondary methylations of mRNA molecules, those on the 2 -hydroxy and the N of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm. 

Poly(A) tails are added to the S end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues. The poly(A) tail appears to protect the S end of mRNA from S —> S exonuclease attack. The presence or absence of the poly(A) tail does not determine whether a precursor molecule in the nucleus appears in the cytoplasm, because all poly(A)-tailed hnRNA molecules do not contribute to cytoplasmic mRNA, nor do all cytoplasmic mRNA molecules contain poly(A) tails... 

Biochemical and genetic experiments in yeast have revealed that the b poly(A) tail and its binding protein, Pablp, are required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. Pablp, bound to the poly(A) tail, interacts with eIF-4G, which in turn binds to eIF-4E that is bound to the cap structure. It is possible that a circular structure is formed and that this helps direct the 40S ribosomal subunit to the b end of the mRNA. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. It appears that a similar mechanism is at work in mammalian cells. 

Figure 39-19. Structure of a typical eukaryotic mRNA showing elements that are involved in regulating mRNA stability. The typical eukaryotic mRNA has a 5 noncoding sequence (5 NCS), a coding region, and a 3 NCS. All are capped at the 5 end, and most have a polyadenylate sequence at the 3 end. The 5 cap and 3 poly(A) tail protect the mRNA against exonuclease attack. Stem-loop structures in the 5 and 3 NCS, features in the coding sequence, and the AU-rich region in the 3 NCS are thought to play roles in mRNA stability.

Interferon is a low molecular weight protein, produced by vims-infected cells, that itself induces the formation of a second protein inhibiting the transcriphon of viral mRNA. Interferon is produced by the host cell in response to the vims particle, the viral nucleic and non-viral agents, including synthetic polynucleides such as polyinosinic acid polycytidylic acid (poly I C). There are two types of interferon. 

Fluorescence measurement using this probe does not require a fluorescence quencher or washing process to suppress the fluorescence emission from nonbinding probes and nonspecific binding probes, which would be advantageous for the detection of mRNAs with poly(A) tracts in cells. 

BBB-RNA must be isolated for molecular cloning and analysis of BBB-specific transcripts, and several methods for isolating BBB-poly (A+) mRNA in one or more steps have been described [68]). Li et al. recently reported that they obtained yields of 12 pg poly (A+) mRNA from a single bovine cortical shell and 3.2 pg poly (A+) mRNA from the pooled cerebral hemispheres of 21 rats [70]. 

The isolation of Poly (A+) mRNA has also led to the synthesis of complementary DNA for the preparation of BBB-cDNA libraries and the construction of BBB-re-... 

The advantage of mRNA over plasmid transfection is the ability of in vitro transcription to allow precise control over features contained within the mRNA (Humphreys et al., 2005 Pillai et al., 2005 Westman et al, 2005). For example, mRNA can be prepared either with or without the physiological m7G(5/)ppp(5/)G cap structure and S poly(A) tail, which are important mediators of canonical translation initiation (Gallie, 1991 Hentze et al., 2006 Iizuka et al, 1994 Kahvejian et al, 2005 Tarun and Sachs, 1995). 

At this stage, mRNA can be polyadenylated using the poly(A) tailing kit (Ambion), according to the manufacturer s instructions. 

Example experiments using the previous methodologies are shown in Fig. 6.1. The major mRNA constructs described in this chapter are dia-grammatically represented in Fig. 6. IB and an example of in vitro transcribed and polyadenylated R-luc-4 sites mRNA is shown in Fig. 6.1A. In these experiments, translation of R-luc-4 sites mRNA is synergistically promoted by the physiological cap structure and the poly (A) tail (Fig. 6.1C), and full miR-dependent translational repression requires the presence of both modifications (Fig. 6.ID, Humphreys etal., 2005). (TheEMCV IRES-containing constructs are discussed later.)... 

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