Brain-specific polyA- transcripts are detected in polyA+ RNA: do complex polyA- brain RNAs really exist?
Transcripts encoded by 2 different rat genomic clones, rg13 and rg100, appear to be typical brain-specific polyA- RNAs, as defined by previous criteria (rare, polysomal, and postnatally expressed from single-copy genes). However, we have found by using a sensitive nuclease protection assay that low levels of these transcripts (10% and 3%, respectively) are detected in polyA+ RNA. To determine if rg transcripts that fractionate as polyA- could have resulted from nicking of polyA+ RNA, we assessed the integrity of 2 known polyA+ RNAs, those of tyrosine hydroxylase, a 2-kilobase (kb) mRNA, and sodium channel, a 9.5-kb RNA. Using RNA prepared by several different procedures, including LiCl-urea and guanidine thiocyanate followed by CsCl centrifugation, the shorter message fractionated as polyA+ after 2 cycles over oligodeoxythymidine (oligo-dT) cellulose, whereas the majority of the longer sodium channel RNA fractionated as polyA, as assayed by nuclease protection using probes from the 5' end of the 2 genes. However, on Northern blots, the same RNA preparations showed an intact 9.5-kb sodium channel band only in polyA+ RNA, suggesting that the polyA- RNAs were randomly cleaved, resulting in a smear of sizes that could not be detected as a discrete band. These data imply that long messages may be nicked during standard isolation procedures and that this would not be detected by Northern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Fung
B P
BP
Neuroscience Program, Tufts University School of Medicine, Boston, Massachusetts 02111.
Brilliant
M H
MH
Chikaraishi
D M
DM
D000375:Aging; D000818:Animals; D001921:Brain / Q000196:embryology / Q000254:growth & development / Q000502:physiology*; D003001:Cloning, Molecular; D005865:Gestational Age; D009928:Organ Specificity; D011061:Poly A / Q000235:genetics*; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D014446:Tyrosine 3-Monooxygenase