Molecular Biology

Chapter 15/16 Outline

 

5¹ cap structure, synthesis and function:

·  The 5¹ end of mRNAs is methylated; The 5¹ end of mRNA can not be labeled using g32P labeled ATP, but can be labeled with b,g32P ATP, suggesting a cap at the 5¹ terminus; The cap is a 7-methylguanosine (m7G) linked to the mRNA through a 5¹ ­ 5¹ triphosphate linkage (F15.3); There are three cap structures, 0, 1 and 2, which differ in 2¹-O-methylation in the first two nucleotides in the mRNA (F15.4); Most eukaryotic mRNAs have cap2.

·  Addition of a 5¹ cap is initiated by removing the g phosphate using RNA triphosphatase, then GTP is added by guanylyl transferase, then methyltransferase adds a methyl group from S-adenosylmethionine to a) the guanine base, the 2¹ hydroxyl of base 1 and the 2¹ hydroxyl of base 2 (F15.5); The cap is added soon after the initiation of transcription because pre-mRNAs of „ 70nt have caps.

·  The 5¹ cap functions to stabilize transcripts; capped viral mRNAs survive injection into Xenopus oocytes much more readily than uncapped mRNAs (F15.7); The 5¹ cap also enhances the translatability of mRNAs as shown using luciferase mRNA (T15.1); The 5¹ cap is also required for transport of mRNA from nucleus to the cytoplasm; By not allowing U1 RNA to be capped by transcribing with Pol III, U1 mRNA is not able to move from nucleus to cytoplasm (F15.8a); The cap is required for removal of the first intron (F15.40); A cap binding complex binds the cap and promotes spliceosome formation.

 

Polyadenylation:

·  mRNAs have polyadenylated (polyA) regions that are ~200nt long (F15.9); The polyAs are on the 3¹ end since it is released quickly by enzymes that degrade RNA from the 3¹ end and base hydrolysis of isolated polyAs yields AMP and one molecule of adenosine (F15.10).

·  The polyA tail protects mRNA from degradation by ~2 fold based on the stability of polyadenylated and deadenylated luciferase mRNA (T15.1); PolyA enhances translatability of mRNA; The polyA tail is bound by polyA binding protein 1 (PAB1), which enhances mRNA translatability; Deadenylated mRNA is translated inefficiently in vitro compared to polyA mRNA (F15.13a); Polyadenylation enhances translation by enabling more efficient recruitment of mRNA into polysomes (F15.14); The effect of polyA on translatability is much larger than its affect on stability (T15.1).

·  The polyA tail is added to the 3¹ end of the mRNA, but only after the mRNA is cleaved (F15.15); ß-globin mRNA made in erythroleukemia cells is transcribed more than 500bp beyond the polyA site, indicating that a cleavage is made before polyadenylation occurs (F15.17); The signal for cleavage of mRNA is an AAUAAA sequence that lies 20-30bp upstream of the cleavage site (F15.18, F15.19); The AAUAAA sequence most effeiciently signals for cleavage when followed by a GU rich then a U rich region starting ~20bp downsteam (F15.20); Placing a synthetic polyA site (SPA) in an exon or an intron  indicates that polyadenylation occurs after mRNAs are at least partially spliced (F15.23).

·  Cleavage of pre-mRNA requires several proteins; Cleavage and polyadenylation specificity factor (CPSF) binds to the AAUAAA and cooperates to promote cleavage stimulating factor (CstF) binding to the GU/U rich region; Cleavage factors I and II and polyA polymerase are also required for cleaving pre-mRNA; The CTD of Pol II is also required with the other cleavage factors to cleave and polyadenylate pre-mRNA (F15.25); Interactions within the precleavage complex (F15.26).  

·  PolyA can be added to a AAUAAA containing RNA oligonucleotide with an optimal distance of 8nt between the AAUAAA and the end of the oligo; Polyadenylation occurs as a slow phase for ~10 As then a rapid addition of ~200 As and the late phase is independent of the AAUAAA (F15.28); The initiation of polyadenylation is dependent upon CPSF binding to the AAUAAA sequence (F15.29a); Elongation of the first 10 As is stimulated by polyA binding protein II (PABII), which binds polyA sequences (F15.30a); Model for polyadenylation (F15.32).

·  PolyA turns over through the action of RNases; Cytoplasmic polyA polymerase can lengthen polyA tails; once polyA tails are gone the RNA will be destroyed; PolyA is required for efficient splicing of the last intron (F15.43).

 

Regulation of mRNA stability:

·  Cytoplasmic mRNAs have widely variable half-lives ranging from 20 minutes to 24hr; Proteins involved in processes that are time restricted often have short mRNA half-lives; Many mRNAs having short half lives contain the sequence AUUUA in the 3¹UTR, which destabilizes mRNA.

·  Casein is a milk protein that is produced in mammary glands after stimulation by prolactin; casein mRNA is stabilized in response to prolactin (T16.1); Measuring half lives of mRNAs by a pulse chase experiment.

·  Regulation stability of the mRNAs encoding transferrin receptor (TfR) iron import protein and ferritin iron storage protein; When cells need more iron they increase TfR and decrease ferritin, and when cells have too much iron the opposite regulation occurs; Regulation of TfR occurs at the level of mRNA stability; Iron dependent stability of TfR and is mediated by iron response elements (IREs) their UTRs (F16.26, F16.27), which are bound by a protein (F16.28); IREs are required to stabilize TfR mRNA (F16.30, F16.31, F16.33); IRE binding protein protects the mRNA from cleavage by RNase, which destabilizes TfR mRNA (F16.36).