Identification of in Vivo mRNA Decay Intermediates Corresponding to Sites of in Vitro Cleavage by Polysomal Ribonuclease 1

Previous work from this laboratory identified a polysome-associated endonuclease whose activation by estrogen correlates with the coordinate destabilization of serum protein mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a novel member of the peroxidase gene family. A characteristic feature of PMR-1 is its ability to generate in vitro degradation intermediates by cleaving within overlapping APyrUGA elements in the 5′-coding region of albumin mRNA. The current study sought to determine whether the in vivo destabilization of albumin mRNA following estrogen administration involves the generation of decay intermediates that could be identified as products of PMR-1 cleavage. A sensitive ligation-mediated polymerase chain reaction technique was developed to identify labile decay intermediates, and its validity in identifying PMR-1-generated decay intermediates of albumin mRNA was confirmed by primer extension experiments performed with liver RNA that was isolated from estrogen-treated frogs or digestedin vitro with the purified endonuclease. Ligation-mediated polymerase chain reaction was also used to identify decay intermediates from the 3′-end of albumin mRNA, and as a final proof of principle it was employed to identify in vivo decay intermediates of the c-myc coding region instability determinant corresponding to sites of in vitro cleavage by a polysome-associated endonuclease. Previous work from this laboratory identified a polysome-associated endonuclease whose activation by estrogen correlates with the coordinate destabilization of serum protein mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a novel member of the peroxidase gene family. A characteristic feature of PMR-1 is its ability to generate in vitro degradation intermediates by cleaving within overlapping APyrUGA elements in the 5′-coding region of albumin mRNA. The current study sought to determine whether the in vivo destabilization of albumin mRNA following estrogen administration involves the generation of decay intermediates that could be identified as products of PMR-1 cleavage. A sensitive ligation-mediated polymerase chain reaction technique was developed to identify labile decay intermediates, and its validity in identifying PMR-1-generated decay intermediates of albumin mRNA was confirmed by primer extension experiments performed with liver RNA that was isolated from estrogen-treated frogs or digestedin vitro with the purified endonuclease. Ligation-mediated polymerase chain reaction was also used to identify decay intermediates from the 3′-end of albumin mRNA, and as a final proof of principle it was employed to identify in vivo decay intermediates of the c-myc coding region instability determinant corresponding to sites of in vitro cleavage by a polysome-associated endonuclease. The process of mRNA decay is closely linked to translation (1Jacobson A. Peltz S.W. Annu. Rev. Biochem... 1996; 65: 693-739Google Scholar), and a growing body of data links the binding of proteins to the mRNA 3′-end with the efficiency of cap-dependent translation initiation (2Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem... 1999; 68: 913-963Google Scholar, 3Imataka H. Gradi A. Sonenberg N. EMBO J... 1998; 17: 7480-7489Google Scholar). mRNA decay in vertebrates can be generally divided into four basic pathways: deadenylation with the possible link to decapping and 5′-3′ degradation of the mRNA body, deadenylation followed by 3′-5′ decay of the mRNA body, nonsense-mediated decay, which may or may not involve decapping, and endonuclease-mediated decay (4Ross J. Microbiol. Rev... 1995; 59: 423-450Google Scholar, 5Schoenberg D.R. Chernokalskaya E. Harford J. Morris D.R. mRNA Metabolism and Post-transcriptional Gene Regulation.Wiley, New York. 1997; : 217-240Google Scholar, 6Tharun S. Parker R. Harford J. Morris D.R. mRNA Metabolism and Post-transcriptional Gene Regulation.Wiley, New York. 1997; : 181-200Google Scholar). All of these effectively serve to disrupt the link between the 5′- and 3′-ends of the mRNA. It has yet to be proven whether the general pathway of mRNA decay in vertebrates will recapitulate processes that have been characterized in yeast, but there is suggestive evidence to support this. A significant number of mRNAs are also degraded through endonuclease-mediated pathways (5Schoenberg D.R. Chernokalskaya E. Harford J. Morris D.R. mRNA Metabolism and Post-transcriptional Gene Regulation.Wiley, New York. 1997; : 217-240Google Scholar). Examples include mRNAs for apo-very low density lipoprotein II (7Binder R. Hwang S.P. Ratnasabapathy R. Williams D.L. J. Biol. Chem... 1989; 264: 16910-16918Google Scholar), c-myc (8Lee C.H. Leeds P. Ross J. J. Biol. Chem... 1998; 273: 25261-25271Google Scholar), transferrin receptor (9Binder R. Horowitz J.A. Basilion J.P. Koeller D.M. Klausner R.D. Harford J.B. EMBO J... 1994; 13: 1969-1980Google Scholar), insulin-like growth factor II (10van Dijk E.L. Sussenbach J.S. Holthuizen P.E. RNA ( N. Y. ).. 1998; 4: 1632-1635Google Scholar), and α-globin (11Wang Z. Kiledjian M. EMBO J... 2000; 19: 295-305Google Scholar). Estrogen induces a global change in the profile of proteins expressed in Xenopus liver resulting from the coordinate destabilization of the serum protein mRNAs (12Pastori R.L. Moskaitis J.E. Buzek S.W. Schoenberg D.R. Mol. Endocrinol... 1991; 5: 461-468Google Scholar) and the transcriptional induction and stabilization of the mRNA for the yolk protein precursor vitellogenin (13Nielsen D.A. Shapiro D.J. Mol. Cell. Biol... 1990; 10: 371-376Google Scholar). mRNA destabilization in this tissue is mediated through the activation of polysomal ribonuclease 1 (PMR-1)1 (14Pastori R.L. Moskaitis J.E. Schoenberg D.R. Biochemistry.. 1991; 30: 10490-10498Google Scholar), a novel endonuclease related to the peroxidase gene family (15Chernokalskaya E. DuBell A.N. Cunningham K.S. Hanson M.N. Dompenciel R.E. Schoenberg D.R. RNA ( N. Y. ).. 1998; 4: 1537-1548Google Scholar). PMR-1 is a component of a large mRNP complex bearing its substrate mRNAs, and mRNA decay is initiated by a 22-fold increase in the unit activity of the polysome-bound enzyme following estrogen stimulation. 2Cunningham, K. S., Hanson, M. N., and Schoenberg, D. R. (2001) Nucleic Acids Res. 29,1156–1162.2Cunningham, K. S., Hanson, M. N., and Schoenberg, D. R. (2001) Nucleic Acids Res. 29,1156–1162. This global change in mRNA decay occurs in the absence of major changes in the amount of cellular PMR-1. The purpose of the present study was to determine whether this increase in unit activity is matched by the appearance of albumin mRNA decay intermediates characteristic of endonuclease cleavage by PMR-1. With the exception of insulin-like growth factor II mRNA, whose unique structure results in a remarkably stable in vivoendonuclease degradation product (10van Dijk E.L. Sussenbach J.S. Holthuizen P.E. RNA ( N. Y. ).. 1998; 4: 1632-1635Google Scholar, 16van Dijk E.L. Sussenbach J.S. Holthuizen P.E. J. Mol. Biol... 2000; 300: 449-467Google Scholar), most mRNAs are degraded without significant accumulation of decay intermediates. However, decay intermediates have been observed using crude in vitro decay systems. The most likely explanation for this is that in vivo these intermediates are subject to rapid exonucleolytic clearance in a manner similar to that which occurs in prokaryotes, where polynucleotide phosphorylase or RNase II rapidly degrade intermediates generated by RNase E (5Schoenberg D.R. Chernokalskaya E. Harford J. Morris D.R. mRNA Metabolism and Post-transcriptional Gene Regulation.Wiley, New York. 1997; : 217-240Google Scholar). For mRNAs that do not show obvious degradation intermediates only a few, highly abundant mRNAs have been examined, in general using primer extension or S1 nuclease protection assays (17Schoenberg D.R. Cunningham K.S. Methods.. 1999; 17: 60-73Google Scholar). Although these techniques do work, they require long exposure times to visualize metastable products and are not readily applicable to low copy mRNAs. We describe here a new approach using ligation-mediated (LM) RT-PCR to identify the 3′-ends of degradation intermediates generated in vivo as a result of either endonuclease cleavage or pausing of a 3′- to 5′-exonuclease. The rationale behind this was our finding that PMR-1 generates degradation intermediates with 3′-hydroxyls, which are good substrates for further degradation by 3′- to 5′-exonucleases, whereas decay intermediates with 3′-phosphate termini are poor substrates for such enzymes. Using this assay we show that the estrogen-induced increase in unit activity of polysome-bound PMR-1 is accompanied by the appearance of albumin mRNA degradation intermediates corresponding to those generated by in vitrocleavage with the purified endonuclease. As a proof of principle this approach was used to demonstrate in vivo cleavage of c-myc mRNA in the coding region determinant that has been identified from in vitro studies as a potential regulatory element by Ross and coworkers (8Lee C.H. Leeds P. Ross J. J. Biol. Chem... 1998; 273: 25261-25271Google Scholar, 18Doyle G.A.R. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. Nucleic Acids Res... 1998; 26: 5036-5044Google Scholar). Male Xenopus were obtained from Xenopus One (Ann Arbor, MI), fed a synthetic diet, and maintained in plastic aquaria with a 12-h light-dark cycle. One milligram of estradiol-17β was injected in 0.1 ml of a solution of 10% Me2SO/90% propylene glycol into the dorsal lymph sac. Animals were anesthetized with 0.1% tricaine methanesulfonate prior to removing the liver. Livers were removed and perfused with ice-cold 1× SSC to remove as much blood as were into followed by the of of tissue of and the was for using a The was for to remove of was and the was for to 0.1 of liver was and the was ml of in a and in a The was and the RNA was in ml of for RNA was with of and with of was to and RNA was by of of ice-cold The RNA was with and in prior to were obtained from were in 10% cellular RNA was isolated using following the and of RNA was to a reaction 1 glycol of ribonuclease and of primer The primer was by and has and were performed for with of RNA The reaction was with of and with of The was to and RNA with the primer was by of of ice-cold The of RNA to the primer was in of followed by of of the primer which is to the of The solution was to for and this was of and to a of The was for followed by the of of II and for The reaction was by for of the reaction was with of 1 this was and to a final of The of the was by the to for and the was of of and of polymerase from were to a final of The was for and was performed for for for and for 1 followed by extension for using by The used of albumin of albumin and of c-myc The reaction were as with and products were by The was in of and of 1 0.1% and 0.1% This was for and into a of a from the reaction were identified by of the The of were from the following a from H. H. R. P. Methods.. 1998; Scholar). The was with the and was used to the four of A was used to and the were in of for The was for with to the This was for and the was was by of of of and of followed by for The was by for with and in of of the was to a reaction of a of 1 The reaction was to followed by the of 1 of primer 1 of the and of polymerase was performed as following which was with of and of for to remove This reaction was by for followed by of of the This was to the by for and for and using polymerase The products were and a as A corresponding to region in of albumin was as (17Schoenberg D.R. Cunningham K.S. Methods.. 1999; 17: 60-73Google Scholar). This was with of from estrogen-treated frogs or of PMR-1 purified as R.E. Schoenberg D.R. J. Biol. Chem... 1995; Scholar, K.S. Hanson M.N. Schoenberg D.R. New York. Scholar) in 1 and One unit of PMR-1 is the amount to of albumin substrate in The were by of solution 0.1% and 0.1% and for The were a and cleavage products were by of decay intermediates in the 3′-end of albumin mRNA. was performed with of liver RNA from a estrogen-treated using a primer that from to the of albumin mRNA. The products were a and the of by were from the and A is in 1 the of the 3′-end of albumin mRNA corresponding to the region in A is with vivo degradation intermediates APyrUGA sites A corresponding to of albumin mRNA was to of from the RNase This was for and to this was of RNase of RNase were for and the reaction was by of of RNase RNA was by of of and of followed by The was in of and products were a and by The of identified by RNase were used to the structure of this of albumin mRNA using the J. M. J. Mol. Biol... 1999; Scholar). extension of albumin mRNA was performed as (17Schoenberg D.R. Cunningham K.S. Methods.. 1999; 17: 60-73Google Scholar). of liver RNA isolated of were either or as with or of purified PMR-1. The were to PMR-1, and was with of primer which is to of albumin mRNA. The RNA and primer were in of 1 for and to was a of and of to a of The reaction was for and by the of of solution 1 were by with of The final were in of and a The of primer extension and nuclease cleavage sites was to a from the and the work that degradation intermediates generated by in vitro PMR-1 cleavage of albumin mRNA 3′-hydroxyls, to degradation by 3′- to E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). We of this to a generally applicable approach to the vivo mRNA degradation intermediates. The in 1 involves of a primer to the 3′-ends of RNA followed by with a primer to generate a of corresponding to of the RNA in the The is using a primer to the mRNA of and the primer used for and the resulting products are a identified by are and to identify the 3′-end of the mRNA degradation the between the primer and the mRNA. to this process is the of a primer that has been by of a for the to the and a to further of the primer This technique not between decay intermediates generated by endonuclease cleavage those by pausing of a 3′- to but as the have not been in mRNAs bearing PMR-1 was identified and purified its ability to generate a unique product by cleaving within overlapping APyrUGA elements in the 5′-coding region of the (14Pastori R.L. Moskaitis J.E. Schoenberg D.R. Biochemistry.. 1991; 30: 10490-10498Google Scholar, R.E. Schoenberg D.R. J. Biol. Chem... 1995; Scholar). Although PMR-1 within APyrUGA it is not a endonuclease and also elements (15Chernokalskaya E. DuBell A.N. Cunningham K.S. Hanson M.N. Dompenciel R.E. Schoenberg D.R. RNA ( N. Y. ).. 1998; 4: 1537-1548Google Scholar, E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). a of albumin mRNA with the of the APyrUGA elements in to the coding and All of these elements within the coding and there are of overlapping elements with the in vitrocleavage of PMR-1 were characterized with the of the 5′-coding identified as A in this was for the appearance of in vivo degradation intermediates following estrogen were injected with 1 of and liver RNA isolated or was by using the primer with a in of albumin mRNA. RNA isolated was used as a A the of RT-PCR products generated by with The the of the to the cleavage sites in structure of the corresponding region of albumin mRNA. A product from albumin mRNA was to in major products from liver RNA were in of the of which to and the of which was to the primer to be identified was the appearance of new products and estrogen administration of the was from the and to identify the between the primer and albumin mRNA degradation of this is in for The to cleavage within the APyrUGA element in the of this of albumin mRNA. The of the in vivo cleavage sites identified in region A are in corresponding to the products in A. The this vivo sites that to the sites of in vitro cleavage by purified PMR-1. Previous work characterized PMR-1 as cleaving between the in the APyrUGA element E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). The of cleavage sites by the of products that this was by a a result that likely from the of and to RNA degradation intermediates. The cleavage between the and a result that is with of cleavage APyrUGA to E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). The cleavage sites are in the the experiments in primer extension was used to the of in vivo decay intermediates and to show that these from cleavage by PMR-1. the in A liver RNA was isolated from frogs or frogs injected with prior to and primer extension was performed using a primer for region A by the the for albumin mRNA in The of the cleavage were to a from albumin using the Although the 12-h liver RNA albumin mRNA, this and RNA for products that from the primer to the the primer extension will mRNA decay intermediates from the of the primer these results that there is a of decay intermediates that are not as products by with the results in there was evidence for decay intermediates within the of albumin mRNA from and RNA from 12-h estrogen-treated frogs the in vivo decay intermediates by primer extension the as those identified in by determine whether these products from in by PMR-1 we the 12-h liver RNA vitro with purified PMR-1. this a significant amount of the of albumin mRNA, we that further in with purified PMR-1 the of products generated by PMR-1 cleavage in these decay intermediates were not generated by PMR-1, vitro with the purified enzyme generate a of primer extension The results of with of purified PMR-1 are in 1 of B. vitro cleavage of RNA that was in vivo in for of the products identified by and primer work that these decay products are unique to PMR-1 E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar) these results that the identified decay intermediates from in by PMR-1. region A in the 5′-coding of albumin mRNA, metastable degradation intermediates were observed in the of in vitro decay experiments using from the 3′-end of albumin mRNA (14Pastori R.L. Moskaitis J.E. Schoenberg D.R. Biochemistry.. 1991; 30: 10490-10498Google Scholar). to of albumin mRNA, of the coding region and the and has four APyrUGA vivo cleavage within this was by in using the RNA from estrogen-treated frogs in It be that the used here and in are only of cleavage of the four APyrUGA products were identified in the in and in the of region are in B. to the 3′-end of albumin mRNA, and of this confirmed the of the in our work M.N. Chernokalskaya E. Schoenberg D.R. Nucleic Acids Res... 1996; Scholar). the sites and in the 5′-coding there are overlapping APyrUGA elements in this of albumin mRNA and However, of the products corresponding to cleavage and the product for cleavage the APyrUGA element were as as that for in A. determine results with region not those with region we the of in vitro cleavage of region of albumin mRNA using in a manner similar to that used to study cleavage within region A E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). the in region was with from estrogen-treated frogs as (14Pastori R.L. Moskaitis J.E. Schoenberg D.R. Biochemistry.. 1991; 30: 10490-10498Google Scholar). intermediates corresponding to the in vivo cleavage sites identified in are the Although were that are for in vitro the was similar to that observed for in vivo decay by This was in using of purified PMR-1 of only major were which a APyrUGA and the results obtained in we that the between the cleavage with and in intermediates either from the of proteins in the that the ability of PMR-1 to this of albumin mRNA or of these are products of in mRNA APyrUGA is present in a it is the for PMR-1 cleavage in albumin (14Pastori R.L. Moskaitis J.E. Schoenberg D.R. Biochemistry.. 1991; 30: 10490-10498Google Scholar) and vitellogenin mRNA K.S. R.E. Shapiro D.J. Schoenberg D.R. S. 2000; PMR-1 is to within the element present in RNA E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). whether the of within APyrUGA sites in region was to RNA a was with of RNase and the identified were used to this to structure with the J. M. J. Mol. Biol... 1999; Scholar). The results of the are in and the structure is in B. this structure the sites by RNase sites are identified as the APyrUGA elements are and the in vivo cleavage sites are with the APyrUGA element and the are in a large The overlapping APyrUGA sites and are in a structure that cleavage by PMR-1 in a manner similar to that observed E. Dompenciel R.E. Schoenberg D.R. Nucleic Acids Res... 1997; Scholar). This for the cleavage observed here in Although the was present in this it was not in this element was and to be vitro by PMR-1. these results within the 3′-end of albumin mRNA its ability to be by PMR-1. the in vitro decay that observed in vivo by Ross and coworkers identified mRNA instability determinant within the coding region of c-myc mRNA coding region determinant D.J. Prokipcak R.D. Ross J. that is a for in vitro cleavage by a polysome-associated endonuclease activity (8Lee C.H. Leeds P. Ross J. J. Biol. Chem... 1998; 273: 25261-25271Google Scholar) and for binding by a protein G.A.R. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. Nucleic Acids Res... 1998; 26: 5036-5044Google Scholar). was the between the and the degradation of c-myc in we to cleavage within the as a of the ability of to labile in vivo decay intermediates from a and mRNA. the in was performed of RNA isolated from using a primer to of c-myc mRNA. A of products is in and the of this of c-myc mRNA the of the identified in vivo mRNA degradation intermediates is in B. As with albumin mRNA, of these sites was by the of the between the primer and the degradation is this to a for in vitro cleavage of c-myc mRNA (8Lee C.H. Leeds P. Ross J. J. Biol. Chem... 1998; 273: 25261-25271Google Scholar), the that the in vivo degradation of c-myc mRNA involves cleavage. these results demonstrate that the approach to vivo mRNA decay intermediates is generally applicable to highly abundant mRNAs and mRNAs With mRNA degradation in vivo is not accompanied by the appearance of stable degradation intermediates. The most likely explanation for this is that in vivo these intermediates are subject to rapid exonucleolytic However, decay intermediates are observed using crude in vitro decay and the between in vivo vitro decay intermediates from the 3′-end of albumin mRNA in and the of such in in mRNA extension and S1 protection assays have been used to identify in vivo endonuclease cleavage of apo-very low density lipoprotein II mRNA (7Binder R. Hwang S.P. Ratnasabapathy R. Williams D.L. J. Biol. Chem... 1989; 264: 16910-16918Google Scholar) and transferrin receptor mRNA (9Binder R. Horowitz J.A. Basilion J.P. Koeller D.M. Klausner R.D. Harford J.B. EMBO J... 1994; 13: 1969-1980Google Scholar), and we have used these to demonstrate in vivo cleavage within the APyrUGA PMR-1 cleavage sites in region A of albumin mRNA here and in a (17Schoenberg D.R. Cunningham K.S. Methods.. 1999; 17: 60-73Google Scholar). However, these techniques work with highly abundant mRNAs, and with these its long exposure times may be to visualize decay intermediates. this we ligation-mediated for the rapid and of in vivo mRNA decay intermediates. The by include its of and ability to identify degradation intermediates from mRNAs such as Although and primer extension were used in this study to the of PMR-1 in the in vivo degradation of albumin mRNA, by between the 3′-end of a decay product generated by endonuclease cleavage pausing for a and coworkers of and of were to demonstrate in vivo pausing as has been observed by Parker and coworkers for mRNA decay Parker R. Scholar). This to that the decay intermediates identified for are the products of endonuclease cleavage. Previous work from our laboratory identified PMR-1 as estrogen-induced endonuclease whose activity with the estrogen-induced destabilization of albumin and serum protein mRNAs (14Pastori R.L. Moskaitis J.E. Schoenberg D.R. Biochemistry.. 1991; 30: 10490-10498Google Scholar). Using to PMR-1 we that this mRNA endonuclease in a that can be with as of a mRNP Estrogen induces a 22-fold increase in unit activity of polysome-bound PMR-1, which is accompanied by the coordinate of albumin mRNA and PMR-1 from The of estrogen-induced in vivo decay intermediates in and corresponding to products of PMR-1 cleavage support to a for PMR-1 in the destabilization of albumin mRNA. We that vitellogenin mRNA, which is and by APyrUGA elements in the that are by PMR-1. The vitellogenin mRNA is by a estrogen-induced protein R.E. Shapiro D.J. J. Biol. Chem... 1997; Scholar), and this binding in vitro cleavage by PMR-1 K.S. R.E. Shapiro D.J. Schoenberg D.R. S. 2000; Scholar). binding by a and albumin mRNA region A the APyrUGA PMR-1 cleavage sites as the vitellogenin mRNA to this highly and is to it from PMR-1 cleavage. here show in vitro and in within the APyrUGA sites in the of albumin mRNA region A within the element in a structure in region these data to the between and structure of mRNA for to cleavage by mRNA endonuclease and for mRNA stabilization resulting from protein binding to these we the degradation of c-myc mRNA as a proof of principle for the of the approach to the of in vivo mRNA decay intermediates. The in the coding region determinant in c-myc mRNA that has been vitro by Ross and coworkers as a for endonuclease and protection from cleavage by the binding of a protein (8Lee C.H. Leeds P. Ross J. J. Biol. Chem... 1998; 273: 25261-25271Google Scholar, 18Doyle G.A.R. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. Nucleic Acids Res... 1998; 26: 5036-5044Google Scholar, R.D. D.J. Ross J. J. Biol. Chem... 1994; Scholar, P. Betz N.A. Ross J. 1997; Scholar), but whose vivo yet to be results identified degradation intermediates in this of which to the major for in vitro cleavage by a polysome-associated endonuclease (8Lee C.H. Leeds P. Ross J. J. Biol. Chem... 1998; 273: 25261-25271Google Scholar). results further validity to the of in vitro for the of mRNA decay and the general of to the of in vivo decay intermediates. We Chernokalskaya for with the primer extension experiments in this polysomal ribonuclease 1 chain reaction ligation-mediated serum albumin