The role of RNA chaperone Hfq in poly(A) metabolism methods to determine positions, abundance, and lengths of short oligo(A) tails.
Polyadenylation is a posttranscriptional modification of RNA occurring in prokaryotes, eukaryotes, and organelles. Long poly(A) tails help export eukaryotic mRNAs and promote mRNA stability and translation, whereas the short bacterial tails facilitate RNA decay. The scarcity of polyadenylated RNAs is one of the obstacles for investigators studying bacterial polyadenylation. The two methods described in this chapter were developed to determine how the poly(A) binding protein Hfq affects the polyadenylation of bacterial RNAs. The first is a 3'-RACE protocol specific to oligoadenylated RNA. This method was designed to rapidly collect a large amount of poly(A) containing 3'-terminal sequences to perform statistical analysis. The second method is an RNA sizing protocol to analyze the polyadenylation status of primary transcripts that were not efficiently detected by 3'-RACE. The latter procedure is based on Northern blot analysis of 3'-RNA fragments generated by RNase H. In the presence of a gene-specific methylated chimeric RNA-DNA oligonucleotide, the enzyme is directed to a unique cleavage site. The 3'-RNA fragments, differing by just one nucleotide at their 3'-ends, are then separated in polyacrylamide gels.
Régnier
Philippe
P
CNRS UPR9073, Paris, France.
Hajnsdorf
Eliane
E
D001483:Base Sequence; D015152:Blotting, Northern; D029968:Escherichia coli Proteins / Q000502:physiology*; D035001:Host Factor 1 Protein / Q000502:physiology*; D018832:Molecular Chaperones / Q000502:physiology*; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D018832:Molecular Chaperones; D012333:RNA, Messenger; D011061:Poly A