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Jolene first appears at the very beginning of Chapter 3, in Grubba's office. She leads Mario and his partners to their locker room, and then leaves. By talking to Bandy Andy, Mario can discover that occasionally, Jolene had been seen going into places (bathrooms and such), but never coming back out.
Mario enters the tournament of Glitzville, and as he advances in rank, some of the fighters start to go missing. He also starts getting E-mails from a strange person called "X". One such E-mail tells him to look in the attic of the building, where he finds the crumpled bodies of fellow fighters King K and Bandy Andy. While exiting the attic, he spies Jolene about to enter, then sees her quickly running out again when she sees him.
However, at the end of the chapter, it is revealed that Grubba is actually behind this. After Mario defeats Macho Grubba, Jolene walks in and tells him that she is actually the sister of the old champion of the Glitz Pit, Prince Mush. Prince Mush disappeared, and Jolene suspected Grubba of kidnapping him, explaining why she got a job as his secretary: so that she could learn his secrets. She also revealed that she was actually "X", and had sent Mario the E-mails so that he would unknowingly help her uncover the mystery, as she thought he would be the only one who could defeat Grubba.
She goes on to question the now-defeated Grubba about her brother, but Grubba simply says that Prince Mush "ain't around these parts no more". Grief-stricken, Jolene is surprised when Prince Mush suddenly appears from the Gold Star Grubba was using to power his machine; Grubba had been using Prince Mush to give the Crystal Star more power. Happily reunited with her brother, Jolene grants Mario the third Crystal Star, so that the tragic events caused by it may never reoccur. She then goes on to become the manager and referee of the Glitz Pit, taking over the Match Reservation System and the rest of Grubba's duties for the remainder of the game.
She appears to be friends with Toodles, as suggested by one of the troubles at the Trouble Center that Mario can solve. Jolene has borrowed the wrestling magazine from Toodles, who wants it back. Jolene also has her own trouble at the Trouble Center, which involves disposing all the garbage in the storeroom by giving it to Goomfrey, whom she has contacted. She says that she could not stand the smell of rubbish and tells Mario to put it somewhere where she could not see it again.
When not at work, Jolene can be found at the Glitzville Fresh Juice Shop; though not stated outright, it is heavily implied throughout the game, mainly from her recognizable appearance. This would also explain her going into bathrooms and not coming out: she actually just changes her clothes and no one recognizes her, although it is also implied that she uses a certain "man-eating toilet" to warp from room to room via the bathroom, like Mario did when a security guard locked him in an abandoned locker room. Also, when X tells Mario to get the Super Hammer from Podler in an e-mail, "Jolene" is not present.
Goombella can use Tattle on Jolene after she escorts Mario to the minor-league locker room of the Glitz Pit but before Mario leaves for first match, when she is inside Glitzville's Fresh Juice Shop without her glasses and suit, and when she is in her Glitz Pit office (having succeeded Grubba).
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