Control of SERCA2a Ca2+ pump mRNA stability by nuclear proteins: role of domains in the 3'-untranslated region.
Alternative splicing of the sarco/endoplasmic reticulum (SERCA2) Ca2+ pump transcript generates the two isoforms: SERCA2a in left ventricular myocytes (LVM) and SERCA2b in most tissues. Nuclear protein extracts from left ventricular myocytes can cause a decay of the 3'-region of the SERCA2a. To determine if all the domains in the 800 b SERCA2a 3'-end region (3344-4243) are equally stable, we examined in vitro decay of synthetically capped, polyadenylated overlapping RNA fragments 2A1-2A6 from the 3'-end region of SERCA2a. Whereas 2A1-2A5 RNAs were stable, the distal fragment 2A6 (4135-4243 b) decayed rapidly. Deleting the 2A6 sequence from the 800-b 3'-end region increased its stability. In mobility shift assays, 2A6 bound to protein(s) in the LVM nuclear extracts in a specific manner: unlabelled 2A6 or the 800 b 3'-region RNA competed for binding but poly A, poly U, and poly C RNA did not. Secondary structure analysis revealed three hairpin loops in 2A6. Experiments using small synthetic RNA fragments for competition with 2A6 binding to nuclear proteins were consistent with a model involving the three hairpin loops. Thus, the secondary structure of the distal domain of SERCA2a RNA may be important in regulating its stability.
Misquitta
Christine M
CM
Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Ghosh
Paromita
P
Mwanjewe
James
J
Grover
Ashok K
AK
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D066298:In Vitro Techniques; D032383:Myocytes, Cardiac / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D020871:RNA Stability / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D038621:Regulatory Sequences, Ribonucleic Acid / Q000502:physiology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D020413:3' Untranslated Regions; D009687:Nuclear Proteins; D012333:RNA, Messenger; D038621:Regulatory Sequences, Ribonucleic Acid; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases